Protective effect of thyroid and restores of ovarian function of Buzhong Yiqi granule (补中益气颗粒) on experimental autoimmune thyroiditis in female rats

Abstract

OBJECTIVE:

To observe the effects of Buzhong Yiqi granule (补中益气颗粒) on thyroid function and ovarian function in rats with experimental autoimmune thyroiditis (EAT).

METHODS:

EAT model was replicate by using the method of mixing and injecting porcine thyroglobulin with Freund's adjuvant and high iodine. Rats were randomly divided into normal control (NC) group, EAT model (EAT) group, selenium yeast (PC) group, low dose Buzhong Yiqi (BZYQ-L) group, medium dose Buzhong Yiqi (BZYQ-M) group and high dose Buzhong Yiqi (BZYQ-H) group. After two months of drug intervention according to dosage, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of free triiodothyronine (FT3), free thyroxine (FT4), thyroid-stimulating hormone (TSH), anti-thyroid peroxidase antibody (TPOAb), thyroglobulin antibody (TGAb) in peripheral blood of rats. The pathological changes of rat thyroid tissues were observed under light microscope with HE staining; ELISA was used to determine estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), anti-müllerian hormone (AMH), and the pathological changes of rat ovarian tissues were observed under light microscope with hematoxylin and eosin staining.

RESULTS:

Compared with the NC group, BZYQ granule improved the thyroid and ovarian tissue morphology, and the levels of TPOAb, TGAb and TSH in the model group rats significantly increased (P < 0.05), the thyroid tissue was severely destroyed, the levels of E2, FSH, LH, T, AMH significantly increased (P < 0.05), and the ovary exhibited polycystic changes; Compared with the model group, TSH level in the BZYQ-L group rats decreased (P < 0.05), FSH, T, AMH levels decreased (P < 0.05), in the BZYQ-M group TPOAb, TSH levels decreased (P < 0.05), FSH, LH, T, AMH levels significantly decreased (P < 0.05), BZYQ-H group TPOAb, TGAb, TSH levels significantly decreased (P < 0.05), FSH, LH, T, AMH levels significantly decreased (P < 0.05), with the greatest improvement and significantly better than selenium yeast group (P < 0.05).

CONCLUSIONS:

BZYQ granule could regulate the thyroid function of EAT rats, reduce thyroid antibody titers, then act on the ovarian function, regulate hormone disorders, and alleviate the pathological damage of rat's ovarian tissues. The effect of high dose Buzhong Yiqi granule is the best.

1. INTRODUCTION

Hashimoto's thyroiditis (HT) is an common chronic lymphocytic or autoimmune thyroiditis that characterized by remarkable high level of autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO), showing effacement of thyroid architecture by diffuse lymphocyte infiltration and residual thyroid follicles antigens.¹ The prevalence of Hashimoto's thyroiditis has risen sharply in the last 30 years.^2,3 In addition, 80% of all diagnosed autoimmune diseases are women.^4,5 More rarely, HT may occur concurrent with other endo-crinopathies or non-endocrine autoimmune diseases. Relationship between autoimmune abnormalities and reproductive failure had been studied extensively,^6,7 and increasing amount evidence links autoimmunity with adverse pregnancy outcomes.⁸ The antibody-mediated autoimmune response through the antibodies against thyroid peroxidase (TPOAbs), which has been suggested as cause of infertility and increased in the risk of miscarriage and preterm birth in pregnant women.^9,⇓-11

There have been a number of studies in recent years evaluating the impact of thyroid autoimmunity on fertility and ovarian reserve. In a secondary analysis of two multicenter randomized controlled trials with 1468 infertile women serum samples, demonstrated that anti-TPO Ab positivity was associated with a doubling of miscarriage risk.¹²

Analysis of a retrospective study of 1495 women clinical series showed that the incidence of thyroid dysfunction was 13.4% in patients with menstrual disturbances or infertility, and the incidence of menstrual disturbances in thyroid dysfunction patients was 1.49 times higher than in normal thyroid functional cases.¹³ Additionally, menstrual disturbances and thyroid dysfunction often interact and coexist.¹⁴ However, the mainstream of treatment of hashimoto's thyroiditis was based on management of the hypothyroidism with a substitution therapy and symptoms control,^3,9 but does not cure the disease, the beneficial effects along the ovarian function still somewhat unclear and needed to be further investigated.

According to Chinese Medicine Formulas (CMFs) theory, different diseases with similar pathogenesis can be treated with one Chinese formula. Buzhong Yiqi decoction (补中益气汤), a famous Chinese’s formula described in the “Treatise on Spleen and Stomach”,¹⁵ have been widely used in clinical treatment of spleen-Qi deficiency related diseases for thousands of years, including internal and gynecological diseases.^{15,⇓,⇓,⇓-19} This formula composed of Huangqi (Astragali Radix), Dangshen (Codonopsis Radix), Baizhu (Atractylodis Macrocephalae Rhizoma Torrefactus), Chenpi (Citri Reticulatae Pericarpium), Shengma (Cimicifugae Rhizoma), Chaihu (Bupleuri Radix Acetatus), Gancao (Glycyrrhizae Radix et Rhizoma), Danggui (Angelicae Sinensis Radix), Shengjiang (Rhizoma Zingiberis Recens), Dazao (Fructus Jujubae).²⁰ Previous studies reveal that Buzhong Yiqi (BZYQ) granule can be used to effectively treat menstrual irregularities and thyroid dysfunction.²¹ It is consistent with the theory of ‘homotherapy for heteropathy’ in Traditional Chinese Medicine (TCM).

However, whether it has a protective influence on HT or ovarian function of the female rats, the molecular mechanism has remained unclear until now. In this study, we aimed to elucidate the protective effect of BZYQ granule on NaI-induced experimental autoimmune thyroiditis (EAT) model in female rats and evaluate the underlying mechanisms.

2. MATERIALS AND METHODS

2.1. Animals

Totally 60 female 6-8-week-old specific pathogen-free (SPF) Sprague-Dawley (SD) rats (body weight, 160-180 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd., License No. SCXK, Beijing, 2019-0010. The rats were housed in the SPF animal laboratory of China-Japan Friendship Hospital [GB14925-2010] under conditions in a temperature-controlled breeding room with a 12 h : 12 h light-dark cycle. All experimental procedures were approved by the Ethics Committee on Animal Care and Use of Beijing China-Japan Friendship Hospital (No. zryhyy 21-21-03-06).

2.2. Drug preparation and chemicals

Buzhong Yiqi granule (SFDA approval number: Z20040120), was provide by Beijing Han Dian Pharmaceutical Co., Ltd. (Beijing, China). Selenious yeast tablets (SFDA approval number: H10940120) was provide by Mudanjiang Lingtai Pharmaceuticals Co., Ltd. (Mudanjiang, China).

Enzyme-linked immunoassay kits, including thyroid peroxidaseantibody (TPOAb, batch number: RX302552R), thyroglobulinantibody (TGAb, batch number: RX302551R), free triiodothyronine (FT3, batch number: RXJ302109R), free thyroxine (FT4, batch number: RXJ302110R), thyroid-stimulating hormone (TSH, batch number: RX302808R), estradiol (E2, batch number: RXJ302812R), follicle-stimulating hormone (FSH, batch number: RX302805R), luteinizing hormone (LH, batch number: RX303076R), testosterone (T, batch number: RXJ302700R), anti-Müllerian hormone (AMH, batch number: RX302471R) were purchased from RuiXin Biotech Co., Ltd. (Quanzhou, China).

2.3. Model establishment and grouping and interventions

After one week of adaptation, the 60 female SD rats were randomly divided into 2 group: the normal control group (NC, sterile distilled water, n = 10) and the EAT model rats (EAT model, distilled water containing 0.05% sodium iodide (NaI, Coolaber, Beijing, China, CS30143404), n = 50).²² An appropriate amount of porcine thyroglobulin (PTg) (Tianfun Xinqu Zhenglong Biochem. Lab, Chengdu, China, 202102) was prepared in a 1 mg/mL solution with phosphate buffer salt (PBS) and then mixed with complete Freund's adjuvant (CFA) (Sigma, St.louis, MO, USA, SLBW7430) or incomplete Freund’s adjuvant (IFA) (Sigma, St.louis, MO, USA, SLCB8702) of equal volume, after which the solutions were fully emulsified into white suspensions to immunize the rats. On the 2nd week and 3rd week, CFA-emulsified PTg (0.1 mL) was subcutaneously injected into the hindlimb foot pads and back of the rats at multiple points.

And then on the 4th-10th weeks, multipoint subcutaneous injections of IFA-emulsified PTg (0.1 mL) were applied to the hind foot pad, the inside of both hind limbs and the back to enhance immunity weekly.

Rats in the normal group were injected with the equivalent volume of PBS in the same places as a control.

At the end of the 10th week, 3 normal rats from NC group and 15 experimental rats from EAT model group were euthanized, and peripheral blood, thyroid was collected for subsequent experiments to confirm if the EAT model established successfully.

Then, the EAT model group was divided into 5 groups: selenious yeast tablets treatment group (positive control, n = 7), BZYQ granule treatment group and the model group (EAT, n = 7). Different treatments were applied the next day after the modeling was completed (after 10 weeks). Selenious yeast tablets were given to the positive control (PC) group 20.83 μg/kg per day. To determine the optimal dosage, different dose of BZYQ granule were administered by medium-dose group (BZYQ-M, 2.13 g/kg, n = 7), high-dose group (BZYQ-H, 4.26 g/kg, n = 7), low-dose group (BZYQ-L, 1.07 g/kg, n = 7). These doses were equivalent to 2 × and 0.5 × the clinical dose.

From the 11th week to the 18th week, the rats in the BZYQ granule treatment group and positive groups were administered the respective drugs once daily. In addition, the rats in NC group and EAT group were orally administered double distilled water until sacrifice. The animal experimental design is shown in Figure 1.

Figure 1. The animal experimental design.

NC: normal control; PC: positive control; EAT: experimental autoimmune thyroiditis; BZYQ-L: low dose Buzhong Yiqi granule; BZYQ-M: medium dose BuzhongYiqi granule; BZYQ-H: high dose BuzhongYiqi granule.

2.4. Sample collection

The day after the last treatment, all the rats were euthanized with 3% pentobarbital sodium after fasting for 12 h. And 5 mL of abdominal aortic blood was collected from each experimental rat and centrifuged at 3000 rpm for 10 min, the upper layer of serum was taken and stored in a freezer at －80 ℃.

2.5. Enzyme-linked immunosorbent assay (ELISA)

Peripheral Blood from rats was centrifuged at 3000 r/min to separate serum for 10 min using a high-speed micro centrifuge (Eppendorf, Hamburg, Germany) at 4 ℃. The concentrations of TPOAb, TGAb, FT3, FT4, TSH；FSH, LH, T, E2, AMH using the corresponding Ellisa kits (Ruixin Biotech, Quanzhou, China) according the manufacturer’s protocol.

2.6. Hematoxylin and eosin (HE) staining

HE staining was used to observe the histopathological of the thyroid and ovarian tissue. Thyroid tissue fixed in 10% paraformaldehyde, embedded in paraffin, and cut into 4-µm-thick sections for routine HE. The morphology of the thyroid tissue was observed under an electron microscope (Nikon Ci-S, Tokyo, Japan) at × 200 magnification and the morphology of the ovarian tissue was at × 40 magnification.

2.7. Statistical analysis

Data are presented as the mean ± standard deviation ($\bar{x}±s$). Statistical analyses were conducted using SPSS software (Version 26.0; IBM Corp., Armonk, NY, USA). Comparisons between groups were conducted using one-way analysis of variance (ANOVA), followed by the least significance difference method (LSD) test for equal variances and Tamhane’s T2 test for unequal variances. Non-normally distributed data were analyzed using the non-parametric test. All statistical tests were conducted by bilateral tests, with a prescribed confidence interval of 95%. A value of P < 0.05 was considered statistically significant.

3. RESULTS

3.1. BZYQ granule alleviated lymphocytic infiltration in thyroids of EAT rats (anatomical observation)

In the normal group, circle or oval thyroid follicles were observed electron microscope, uniform presence of light red colloids in thyroid follicles was abundant with homogeneous distribution, and intact epithelial cells (Figure 2A). EAT rat model was induced by feeding the animal with 0.05% NaI water for 10 weeks, thyroid histology showed a massive number of lymphocytes infiltrated the thyroid gland, thyroid follicles atrophy and disappear and uneven size, irregular morphology and destruction of thyroid follicles in EAT rats with fibrotic tissue presence (Figure 2B). These phenomena indicated that the occurrence of EAT.

Figure 2. Pathological changes in thyroid tissue.

A: thyroid tissue of normal control group (× 200); B: thyroid tissue of EAT group (× 200); C: thyroid tissue of positive control group (× 200); D: thyroid tissue of BZYQ-L group (× 200); E: thyroid tissue of BZYQ-M group (× 200); F: thyroid tissue of BZYQ-H group (× 200). EAT: experimental autoimmune thyroiditis; BZYQ-L: low dose Buzhong Yiqi granule; BZYQ-M: medium dose BuzhongYiqi granule; BZYQ-H: high dose BuzhongYiqi granule. From the 11th week to the 18th week, selenious yeast tablets were given to the positive control group 20.83 μg/kg per day. Different dose of BZYQ granule were administered by medium-dose group (BZYQ-M, 2.13 g/kg, n = 7), high-dose group (BZYQ-H, 4.26 g/kg, n = 7), low-dose group (BZYQ-L, 1.07 g/kg, n = 7) per day. In addition, the rats in NC group and EAT group were orally administered double distilled water until sacrifice. The dyeing method of all pictures is hematoxylin-eosin staining.

Compared with the model group, lymphocytes infiltration was observed in the stroma of the thyroid follicles, only a small subset of follicular structure was destroyed in the PC group (Figure 2C) and BZYQ-L group (Figure 2D). Supplementation with BZYQ granule in a dose-dependent manner, the infiltration of lymphocytes decreased significantly and tissue fibrosis reduced in BZYQ-M treatment groups (Figure 2E), BZYQ-H alleviated lymphocytic infiltration of the thyroid, histology closer to that of NC group (Figure 2F).

3.2. Effect of BZYQ granule downregulated of thyroid serum antibody in EAT rats

The concentration of TPOAb and TGAb in EAT rats elevated significantly compared with the NC group (P < 0.001). Compared with the model group, the concentrations of TPOAb in BZYQ-M (P < 0.05), BZYQ-H (P < 0.05) levels decreased significantly. The levels of TGAb in BZYQ-H (P < 0.001) decreased significantly.

Compared with PC group, the levels of TPOAb in BZYFQ-H group were significantly decreased (P < 0.05). The levels of TGAb in BZYFQ-M group, BZYQ-H group were significantly decreased (P < 0.05) (Table 1).

Table 1.

Thyroid serum antibody in rats (IU/mL, $\bar{x}±s$)

Group	n	TPOAb	TGAb
Normal control	6	0.34±0.09	8.09±2.97
EAT model	7	1.64±0.57a	78.30±23.89a
Positive control	6	1.12±0.15	84.20±9.70
BZYQ-L	7	1.07±0.57	66.67±45.84
BZYQ-M	7	0.86±0.44b	51.86±31.86c
BZYQ-H	7	0.48±0.08bc	16.97±12.32bc
F value		9.693	9.447
P value		< 0.001	< 0.001

Notes: TPOAb: anti-thyroid peroxidase antibody; TGAb: thyroglobulin antibody; EAT: experimental autoimmune thyroiditis; BZYQ-L: low dose Buzhong Yiqi granule; BZYQ-M: medium dose BuzhongYiqi granule; BZYQ-H: high dose BuzhongYiqi granule. From the 11th week to the 18th week, selenious yeast tablets were given to the positive control group 20.83 μg/kg per day. Different dose of BZYQ granule were administered by medium-dose group (BZYQ-M, 2.13 g/kg, n = 7), high-dose group (BZYQ-H, 4.26 g/kg, n = 7), low-dose group (BZYQ-L, 1.07 g/kg, n = 7) per day. In addition, the rats in NC group and EAT group were orally administered double distilled water until sacrifice. One rat in normal control group and one rat in positive control group died during the experiment with open wounds which were considered caused by violent fight among rats. Comparisons between groups were conducted using one-way analysis of variance, followed by the least significance difference method test for equal variances and Tamhane’s T2 test for unequal variances. All statistical tests were conducted by bilateral tests, with a prescribed confidence interval of 95%. A value of P < 0.05 was considered statistically significant. Compared with the normal control group, ^aP < 0.05; compared with EAT model group, ^bP < 0.05; compared with positive control group, ^cP < 0.05.

3.3. Effect of BZYQ granule regulated thyroid function levels in EAT rats

We measured serum TSH, FT3 and FT4 levels to evaluate thyroid function in rats.

Compared with the NC group, the serum TSH concentration in the EAT group was increased (P < 0.01); the serum TSH level in BZYQ granule group were all decreased (P < 0.05), notably, the expression pattern of these hormone was altered in a dose-dependent manner by BZYQ granule treatment, resulting in expression levels closer to that of normal control group rats than EAT group with increasing BZYQ granule dose (Table 2).

Table 2.

Thyroid Function Levels in rats ($\bar{x}±s$)

Group	n	TSH (mU/L)	FT3 (pmol/L)	FT4 (pmol/L)
Normal control	6	0.60±0.33	3.63±3.40	8.85±5.13
EAT model	7	5.39±2.88a	3.24±0.79	17.11±9.93
Positive control	6	2.53±1.70	5.81±3.76	14.24±5.77
BZYQ-L	7	2.35±2.05b	3.36±1.22	14.86±4.79
BZYQ-M	7	1.99±0.95b	2.69±1.05	13.14±5.62
BZYQ-H	7	1.85±1.15b	2.75±0.83	13.45±6.13
F value		5.588	1.851	1.115
P value		0.001	0.129	0.815

Notes: TSH: thyroid-stimulating hormone; FT3: free triiodothyronine; FT4: free thyroxine; EAT: experimental autoimmune thyroiditis. BZYQ-L: low dose Buzhong Yiqi granule. BZYQ-M: medium dose BuzhongYiqi granule. BZYQ-H: high dose BuzhongYiqi granule. Different dose of BZYQ granule were administered by medium-dose group (BZYQ-M, 2.13 g/kg, n = 7), high-dose group (BZYQ-H, 4.26 g/kg, n = 7), low-dose group (BZYQ-L, 1.07 g/kg, n = 7) per day. In addition, the rats in NC group and EAT group were orally administered double distilled water until sacrifice.One rat in normal control group and one rat in positive control group died during the experiment with open wounds which were considered caused by violent fight among rats. Comparisons between groups were conducted using one-way analysis of variance, followed by the least significance difference method test for equal variances and Tamhane’s T2 test for unequal variances. All statistical tests were conducted by bilateral tests, with a prescribed confidence interval of 95%. A value of P < 0.05 was considered statistically significant. Compared with the normal control group, ^aP < 0.05; compared with EAT model group, ^bP < 0.05. From the 11th week to the 18th week, selenious yeast tablets were given to the positive control group 20.83 μg/kg per day.

3.4. Effect of BZYQ granule on the ovarian morphologic changes in EAT model rats

The normal control group rats exhibited ovarian follicles at various normal stages of development and they contained corpora lutea upon HE staining (Figure 3A). However, the EAT model rats ovarian cortex has become thickened notably, multiple cystic dilatated follicles in the ovarian. Additionally, the ovarian morphology disordered with oocytes and corona radiata within these follicles were absent, granular cells arranged loosely and the number of granulosa cell layers markedly reduced, as well as rare corpora lutea (Figure 3B), suggesting a reduction in ovarian reserve and abnormal ovulation in EAT rats.

Figure 3. Ovarian morphology and follicular development.

A: ovary tissue of normal control group (× 40); B: ovary tissue of EAT group (× 40); C: ovary tissue of positive control group (× 40); D: ovary tissue of BZYQ-L group (× 40); E: ovary tissue of BZYQ-M group (× 40); F: Ovary tissue of BZYQ-H group (× 40). EAT: experimental autoimmune thyroiditis; BZYQ-L: low dose Buzhong Yiqi granule; BZYQ-M: medium dose BuzhongYiqi granule; BZYQ-H: high dose BuzhongYiqi granule. From the 11th week to the 18th week, selenious yeast tablets were given to the positive control group 20.83 μg/kg per day. Different dose of BZYQ granule were administered by medium-dose group (BZYQ-M, 2.13 g/kg, n = 7), high-dose group (BZYQ-H, 4.26 g/kg, n = 7), low-dose group (BZYQ-L, 1.07 g/kg, n = 7) per day. In addition, the rats in NC group and EAT group were orally administered double distilled water until sacrifice. The dyeing method of all pictures is hematoxylin-eosin staining.

No significant changes were observed in EAT model group and PC group, except that a slight increase of healthy ovarian follicle numbers was observed in PC group (Figure 3C). An increasing number of granulosa cell layers were observed in BZYQ-L group compared to PC group (Figure 3D). Recovery in regular morphology was observed in BZYQ-M group, in addition, increase in health follicles and corpora lutea (Figure 3E). In the BZYQ-H group, normal tissue morphology was partially restored, with oocytes evident in follicles and an increase in the number of granulosa cell layers (Figure 3F).

3.5. BZYQ granule downregulated serum AMH levels in EAT rats

The AMH level in normal control group was (818 ± 85) pg/mL. The AMH level in EAT model group was (2849 ± 771) pg/mL. In the positive control group, the number went to (1635 ± 488) pg/mL. And in BZYQ treatment groups, the AMH levels were (1017 ± 626) pg/mL in low-dose group, (1293 ± 779) pg/mL in medium-dose group and (845 ± 352) pg/mL in high-dose group. The F value was 11.159 and P < 0.001. EAT model group serum AMH values were increased prominently compared with NC group rats (P < 0.05). A reduction in serum expression was observed in PC group and BZYQ granule treatment group compared to EAT model group. Notably, the expression pattern of AMH was altered in a dose-dependent manner by BZYQ granule treatment, resulting in expression levels closer to that of NC rats, moreover, BZYQ-H reduced AMH expression in EAT model serum compared with PC group (P < 0.05).

3.6. BZYQ granule regulated serum sex hormone levels in EAT rats

As well as these alterations, BZYQ granule of orally administrated animal also displayed a strikingly altered the E₂, FSH, LH and T levels in serum. As presented in Table 3, compared to normal rats, the EAT model rats had elevated serum E₂, FSH, LH and T (P < 0.05). Compared to EAT model rats, the reduced the levels of FSH in PC group (P < 0.05). Significantly different compared with EAT model group rats after the BZYQ granule treatment, the levels of serum FSH, LH, T were significantly lower in BZYQ-M and BZYQ-H group (P < 0.05). Compared to PC group rats, the serum LH and T were reduction remarkably in BZYQ-M (P < 0.05), FSH and T decreased significantly in BZYQ-H group (P < 0.05). These concentrations were reduced in a dose-dependent manner by BZYQ granule.

Table 3.

Serum sex hormone levels in rats ($\bar{x}±s$)

Group	n	E2 (pmol/L)	FSH (mIU/mL)	LH (mIU/mL)	T (ng/mL)
Normal control	6	2.18±1.34	0.05±0.05	2.58±0.32	6.54±3.39
EAT model	7	4.80±2.36a	0.82±0.26a	3.56±0.69a	45.74±17.15a
Positive control	6	3.68±1.73	0.44±0.18b	3.01±0.49	33.98±14.85
BZYQ-L	7	3.23±0.92	0.23±0.16b	2.68±0.96	26.08±6.73b
BZYQ-M	7	3.16±1.47	0.28±0.21b	2.43±0.29bc	15.36±5.34bc
BZYQ-H	7	2.91±2.20	0.21±0.12bc	2.28±0.32bc	18.21±6.82bc
F value		1.015	13.335	3.946	6.531
P value		0.424	< 0.001	0.007	< 0.001

Notes: E_2: estradiol; FSH: follicle-stimulating hormone; LH: luteinizing hormone; T: testosterone; EAT: experimental autoimmune thyroiditis; BZYQ-L: low dose Buzhong Yiqi granule; BZYQ-M: medium dose BuzhongYiqi granule; BZYQ-H: high dose BuzhongYiqi granule. From the 11th week to the 18th week, selenious yeast tablets were given to the positive control group 20.83 μg/kg per day. Different dose of BZYQ granule were administered by medium-dose group (BZYQ-M, 2.13 g/kg, n = 7), high-dose group (BZYQ-H, 4.26 g/kg, n = 7), low-dose group (BZYQ-L, 1.07 g/kg, n = 7) per day. In addition, the rats in NC group and EAT group were orally administered double distilled water until sacrifice.One rat in normal control group and one rat in positive control group died during the experiment with open wounds which were considered caused by violent fight among rats. Comparisons between groups were conducted using one-way analysis of variance, followed by the least significance difference method test for equal variances and Tamhane’s T2 test for unequal variances. All statistical tests were conducted by bilateral tests, with a prescribed confidence interval of 95%. A value of P < 0.05 was considered statistically significant. Compared with the normal control group, ^aP < 0.05; compared with EAT model group, ^bP < 0.05; compared with positive control group, ^cP < 0.05.

4. DISCUSSION

HT was known as chronic lymphocytic thyroiditis, which was the most common autoimmune disease. In this study, Tg+NaI-immunized SD rats were used as experimental models to explore the effects of BZYQ granule on HT and associated mechanisms in vivo. Since HT was predominant in women, female SD rats were selected for modeling in this study. Serologic results showed that the levels of serum TPOAb and TGAb were significantly higher in the EAT model group that in the NC group, in addition, morphological observation of randomly selected model rats showed that a large number of follicular lumens in the thyroid tissue of the EAT model group were destroyed. This EAT animal model was of methods had been used to study human HT.^{23,⇓,⇓-26} These studies have shown consistent serological and pathological results with the present study, with elevated thyroid autoantibodies, thyroid follicular destruction, and significant infiltration of lymphocytes in the thyroid gland being the key indicators of a successful model.

Compared with the normal group, the serum levels of TSH were significantly increased, the levels of FT4 increased and TF3 were decreased in the EAT model group but the difference was not statistically significant, indicating the state was in the subclinical hypothyroidism stage, equivalent to the middle stage of HT. There is usually a long latency period before hypothyroidism occurs.²⁷ Therefore, early intervention may theoretically prevent the development of the disease and maintain the normal structure and thyroid function. Serum hormone levels of FT4 and thyroid antibodies TgAb, TPOAb were slightly decreased in the PC group and BZYQ-L group compared with the EAT model group, but the difference was not statistically significant, indicating that BZYQ granule concentrations in these two groups were not sufficient to treat EAT. In contrast, serologic tests in the high-dose group revealed a significant decreased in thyroid antibodies and TSH compared to the EAT model group. Pathological results also showed that the high-dose group had less lymphocyte infiltration in the interstitial thyroid follicles and a more intact thyroid structure. Despite the selenium was considered as therapy to slow disease progression in subclinical hypothyroidism,²⁸ the evidence quality was deemed low.²⁹ The levels of thyroid autoantibodies are correlated with symptoms such as depression and reduced quality of life in HT patients. However, the primary goal of Western medicine treatment is to control thyroid dysfunction, and even after restoration of thyroid function, some patients may still experience symptoms.²⁸ Selenium is an essential trace element in the human body, and research indicates that selenium deficiency is one of the risk factors for HT.³⁰ Supplementation of selenium is considered to lower thyroid autoantibodies, alleviate thyroid damage, and can be used as an adjunct therapy for HT.³¹ Studies by Qu et al ³¹ have found that oral supplementation of selenium yeast can reduce anti-thyroid antibodies levels in HT patients and regulate inflammatory factors such as interferon-γ (IFN-γ), interleukin-4 (IL-4), as well as oxidative stress markers malondialdehyde, glutathione peroxidase, and superoxide dismutase. Similarly, in experiments, selenium yeast has been shown to lower FT3, FT4, TSH, TGAb, TPOAb, IFN-γ levels and increase IL-4 levels in EAT rats.³² It regulates the immune system and improves thyroid follicle structural damage, lymphocyte infiltration, and interstitial fibrosis in rat thyroid. Therefore, in this experiment, selenious yeast tablets were chosen as the positive control drug. The results showed that BZYQ granule could decreased the thyroid antibodies level and regulated the thyroid function, indicating that BZYQ granule may have a potential therapeutic effect on EAT in female SD rats. It is important to note that the TSH, FT3, FT4 of model group vary in different studies.^26,33 Therefore, further research is necessary to follow-up the thyroid function for long time.

Our data suggested that BZYQ granule may protect ovarian function by reduce the TPOAb, TGAb and TSH, regulate the autoimmune diseases. The disordered ovarian morphology and serum AMH, E₂, FSH, LH and T concentrations were significantly increased in EAT model as observed in this study. AMH belongs to the transforming growth factor β family. It is a homodimeric glycoprotein secreted by granulosa cells of pre-antral and early antral ovarian follicles. A recent Meta-analysis of four case-control and retrospective studies using the AMH evaluated ovarian reserve by Hasegawa et al ³⁴ showed that thyroid autoimmunity and hypothyroidism may affect the ovarian reserve. Similarly, a recent animal model of chronic hypothyroidism studied its effects on the growing follicle population, the substantial evidence in animals that TSH reduction in serum levels leads to reproductive problems.³⁵ In a clinical context, the elevation of FSH and E₂ in the early follicular phase signifies a decline in ovarian reserve, with the decrease in E₂ often occurring after the elevation of FSH, indicating that ovarian function may have significantly deteriorated by this point. The serum AMH level is a biomarker for ovarian reserve and a prognostic marker for fertility.³⁶ In the process of reproductive system development, the AMH is a regulator of ovarian folliculogenesis,³⁷ the AMH excess inhibits the synthesis of LH receptors on granulosa cells and the production of progesterone. It was involved in the regulation of oocyte development and meiosis, inhibiting oocyte maturation. It also inhibits aromatase expression, which leads to a decrease in granulosa cell sensitivity to FSH.³⁸ Besides, excessive secretion of androgens leads to the abnormal initiation of early recruitment, resulting in an excess of small antral follicles, which may contribute to premature arrest of follicles.³⁹

Thyroid hormones influence the metabolism of tissues within the body, as well as the rate of chemical reactions. The endocrine functions of both the thyroid and the gonads are regulated and controlled by neurotransmitters and neurohormones from the hypothalamus, acting through the anterior pituitary. Normal thyroid function is crucial for promoting female reproductive physiology and endocrine function. There is a mutual regulatory interaction between the hypothalamus-pituitary-thyroid (HPT) axis and the hypothalamus-pituitary-ovary (HPO) axis. Changes in thyroid function can impact the secretion of sex hormones and ovulatory function in the ovaries, leading to specific histological characteristics in ovarian morphology.⁴⁰

In this study, the feasibility and mechanism of BZYQ for preventing the ovarian dysfunction that associated with hypothyroidism is investigated based on the EAT model rats. However, the relevant reviews or medical records of Traditional Chinese medicine for the treatment of thyroid function dated back to 2000 before, and many valuable experiences have been accumulated in this area. Therefore, TCM has certain advantages in the treatment of thyroid function, and it is of particular importance to clarify its mechanisms of action in both thyroid function and ovarian function. As regarding to Hashimoto's thyroiditis that was closely related to the physiological function of the “Spleen”, therefore, addressing the dysfunction of spleen should be the primary focus throughout the treatment.⁴¹ For treating HT, professor Xia believes that the spleen functions well in transportation and transformation, abundant Qi and blood generation can be guaranteed, which is based on the accumulating experience in the diagnosis and treatment.⁴² The classic formula Buzhong Yiqi decoction, is a well-established prescription for invigorating the spleen and replenishing Qi. Other studies have found that modified BZYQ decoction has a good effect in the early and middle stages of HT, especially when combined with levothyroxine sodium in the later stages of the disease.⁴³ Wei’s clinical research has shown that BZYQ decoction can improve antibody levels and clinical symptoms in Hashimoto's thyroiditis patients in the short term. In the long run, it can also maintain TGAb and TPOAb at lower levels.⁴⁴

In the TCM theory, the autoimmunity thyroid disease was closely related to the physiological function of the “Spleen”. When the female of childbearing age has hypothyroidism, the clinical symptoms such as rare menstruation, irregular menstruation and endless dripping, abortion, infertility will occur, similar to the spleen deficiency syndrome treated with TCM. Female with HT and ovarian dysfunction often have spleen deficiency symptoms, thus the method of invigorating spleen and nourishing Qi was used. Buzhong Yiqi decoction is one of the spleen-invigorating traditional classic CMF with remarkable impact on recovering the thyroid function and irregular menstruation. HPT and HPO regulation may be one of the mechanisms involved in the treatment of spleen deficiency syndrome. Buzhong Yiqi granule have been proven to have ovarian-protectives effects, and the way they exert their effects is related to regulate HPT and HPO.

In conclusion, HT was a complicated syndrome that appears to lead to infertility. However, there was no single drug that could treat endocrine and reproductive aspects and all the clinical manifestations in between. The Chinese herbal medicine involves the use of several herbs in a formula to ameliorate a set of problems, or a syndrome. Therefore, the use of CHM may be appropriate in disorders such as both hypothyroidism and ovarian dysfunction are at play. This study found that TPOAb and TGAb antibodies were elevated in EAT rats, along with thyroid pathological changes. Thyroid function presented a subclinical hypothyroidism state. Simultaneously, there were disruptions in sex hormones, indicating an initial decline in ovarian function. Observational markers such as AMH and pathology suggested an approximation to polycystic ovary syndrome. BZYQ granule could decrease antibody levels, alleviate subclinical hypothyroidism and thyroid pathology, regulate the reproductive endocrine system, and improve ovarian function, with the high-dose group showing the best results. This research provides additional evidence for the mechanisms of how HT affects ovarian function, and lays the scientific foundation for the clinical application of Traditional Chinese Medicine in treating HT with menstrual irregularities and other conditions under the concept of ‘treating different diseases with the same treatment’.

5. ACKNOWLEDGMENT

We would like to express our deepest gratitude to all those who have provided their support and assistance in the completion of this project.