Intervention and immune regulation of Fuzheng Xiaoying Decoction on experimental autoimmune thyroiditis model rats

QIAO Jia-jun, ZHANG Yu-yang, SHI Jing-ping, XIA Zhong-yuan

1. Beijing University of Chinese Medicine, Beijing 100029, China

2. Department of Chinese Medicine Surgery, China-Japan Friendship Hospital, Beijing 100029, China

Keywords:

ABSTRACT Objective: To investigate the effect of Fuzheng Xiaoying Decoction on experimental autoimmune thyroiditis (EAT) model rats and explore its possible mechanism of immune regulation. Methods: Among the 40 female SD rats, 10 were randomly selected as the blank group, and the rest were immunized with pig thyroglobulin and Freund's adjuvant combined with high iodine feeding to make the EAT model. After the model was established, they were randomly divided into three groups: the model group, the selenium yeast tablet group and the Fuzheng Xiaoying Decoction group, which were continuously gavaged for 2 months.The anti thyroid autoantibodies, thyroid function, IL-38 and Th17/Treg distribution were measured. The pathological changes of thyroid tissue were observed by HE staining. Results:Compared with the blank group, the levels of TPOAb and TGAb in the model group were significantly higher(P＜0.05). Lymphocyte infiltration was seen in the thyroid tissue of the model group,and also the thyroid follicles were partially destroyed, the shape was irregular, and the colloid distribution was uneven, indicating that the modeling was successful. Compared with the model group, the levels of TPOAb and TGAb in the selenium yeast tablet group and the Fuzheng Xiaoying Decoction group decreased significantly(P＜0.05). And the Fuzheng Xiaoying Decoction group had more significant improvement in thyroid follicle destruction and lymphocyte infiltration than the selenium yeast tablet group (P＜0.05). Compared with the blank group, the levels of T3, T4, FT3 and FT4 in the model group increased, the level of IL-38 decreased, the proportion of Th17 increased, and the ratio of Th17/Treg increased(P＜0.05). Compared with the model group, T4 level decreased, IL-38 level increased in the selenium yeast tablet group and the Fuzheng Xiaoying Decoction group(P＜0.05).But there was no statistically significant difference in the reduction of Th17 proportion and Th17/Treg ratio between the above two groups and the model group (P＞0.05). There was no significant difference in Treg ratio among the groups. Conclusion: Fuzheng Xiaoying Decoction can significantly reduce the level of anti thyroid autoantibodies in EAT rats by regulating the immunity of EAT rats, and improve the follicular destruction and lymphocyte infiltration of thyroid tissue in rats.

1. Introduction

Hashimoto's thyroiditis (HT) takes the positive Anti thyroid antibody as the main clinical diagnostic standard. The prevalence rate of positive Anti thyroid antibody in Chinese adult population is 14.19%, ranking the second among thyroid diseases [1]. This disease is easy to be complicated with abnormal thyroid function, especially the main cause of hypothyroidism. At the same time, the increase of autoantibodies is related to infertility or female adverse pregnancy outcomes. The pathogenesis is still unclear, and autoimmune factors account for one of the main factors. The imbalance of Th17-Treg cells is a hot topic in recent years. As a member of the interleukin-1 family, IL-38 can exert anti-inflammatory properties,inhibit Th17 maturation, and regulate Th17-Treg balance [2].Existing studies have shown that selenium supplementation, such as taking selenium yeast tablets, can reduce Anti thyroid antibodies and reduce thyroid damage, and can be used as an adjuvant therapy for HT [3]. We believe that the key pathogenesis for the occurrence and development of HT is based on deficiency and excess, and the main treatment principle is to strengthen the body and eliminate galls[4]. Professor Xia Zhongyuan's Fuzheng Xiaoying Decoction is an empirical formula commonly used in the clinical treatment of HT. In this experiment, Fuzheng Xiaoying Decoction was used as the drug of the experimental group. Through the positive control of selenium yeast tablets, the effects of Fuzheng Xiaoying Decoction on reducing Anti thyroid antibody, improving thyroid function and histopathological damage in EAT rats, as well as on IL-38 level and Th17-Treg balance were observed, so as to provide experimental research basis for the clinical application of Fuzheng Xiaoying Decoction.

2. Material

2.1 Experimental Animal

40 SPF grade female Sprague Dawley rats aged 6-8 weeks,weighing 160-180 g, were purchased from sbefore (scxk (Beijing)2019-0010); They were raised in the SPF level small animal laboratory of the Clinical Research Institute of China Japan Friendship Hospital (in accordance with the national standard gb14925-2010).

2.2 Ethical Review

This experiment has passed the experimental animal ethics of China Japan Friendship Hospital(No.zryhyy21-21-03-06).

2.3 Preparation of Traditional Chinese Medicine

Fuzheng Xiaoying Decoction is composed of:Huangqi 20 g、Chuanshanlong 15 g、Baizhu 15 g、Banxia 10 g、Xiakucao 12 g、Danggui 10 g、Xuanshen 15 g、Tubeimu 15 g、Gancao 3 g etc. Raw materials were purchased from Sinopharm Group Beijing Huamiao Pharmaceutical Co., Ltd. and Beijing meikangtang Pharmaceutical Technology Co., Ltd. After the adult is set to 60kg,the crude drug amount is converted according to the conversion coefficient of the heavy dose of rats and human body in 6.2. The above Chinese herbal pieces are decocted twice, filtered and combined, concentrated into a crude drug amount of 1.715 g/mL,and then packaged and stored in cold storage after high-temperature and high-pressure sterilization.

2.4 Drugs and Reagents

Selenium yeast tablets (trade name: siver, batch number: 210204),porcine thyroglobulin (PTg, Huayang Zhenglong biochemical products research laboratory, Tianfu New District, batch number:202102); Complete Freund's Adjuvant (CFA, sigma, USA, batch No.:slbw7430), Incomplete Freund's Adjuvant (IFA, sigma, USA, batch No.: slcb8702); Sodium iodide (NaI, Beijing Kulaibo Technology Co., Ltd., batch No.: cs30143404); Thyroglobulin antibody (TGAb),Free Triiodothyronine (FT3), Triiodothyronine (T3), Free Thyroxine(FT4), Thyroxine (T4), Thyroid Stimulating Hormone (TSH),Thyroid Peroxidase antibody (TPOAb) Interleukin-38 (IL-38)ELISA Kit (Jiangsu Enzyme Immunoassay Industry Co., Ltd., batch numbers: 20210922-0582, 20210922-70747, 20210922-20356,20210922-0584, 20210922-0573, 20210922-20215, 20210922-0519,220415-71280r1); Pacific Blue anti-rat CD45 Antibody (Biolegend,USA, Cat# 202225); FITC anti-rat CD3 Antibody(Biolegend, USA,Cat# 201403); CD8 PerCP(Biolegend, USA, Cat# 201712); PE-Cy7 Mouse Anti-Rat CD4(BD, USA, Cat#561578); CD25 PE(Thermo Fisher,USA, Cat#12-0390-82); FOXP3 APC(Thermo Fisher,USA,Cat#17-5773-82); Rat IgG2a(Thermo Fisher,USA, Cat#12-4321-80/17-4321-81); IL-17A PE(Thermo Fisher,USA, Cat#12-7177-81).

2.5 Instrument

An upright optical microscope BX-53 (Olympus); Enzyme label analyzer RT-6100 (rayto),; 37 ℃, 5% CO2incubator (Thermo Fisher); Flow cytometry FACS (Becton Dickinson).

3. Method

3.1 Build EAT model

10 SD rats were selected as the blank group by random number table method. The other rats were given high iodine drinking water containing 0.05% NaI and mixed immunization with Freund's adjuvant and PTg. They were divided into primary immunization for 1 week and booster immunization for 6 weeks. Each rat was injected with 100 μg PTg. The EAT model was made and randomly divided into three groups: model group, selenium yeast tablet group and Fuzheng Xiaoying Decoction group. Refer to the previous research of this research group for modeling methods [5, 6]. The blank group was given drinking water at the same time, and 0.2 mL PBS buffer was injected subcutaneously each time. The increase of TGAb and TPOAb in the serum of rats after modeling is consistent with the clinical diagnosis of the disease. The infiltration of lymphocytes and plasma cells in the thyroid tissue, reduced glia, shrinking and even destruction of follicles, and fibrosis in the later stage are consistent with the pathological diagnosis of the disease[7].

3.2 Grouping and Administration

After successful modeling, rats in Fuzheng Xiaoying Decoction group were given 10 mL·kg-1·d-1Fuzheng Xiaoying Decoction; The selenium yeast tablet group was given 20.67 μg·kg-1·d-1selenium yeast tablets [according to adult dose 3.33 μg·kg-1·d-1calculation];The rats in the blank group and the model group were given 10 mL·kg-1·d-1double distilled water by gavage.The body weight was measured and the dosage was adjusted according to the body weight.The course of treatment was 2 months.After the last administration,the rats in each group fasted for 12 hours and then killed.

3.3 Testing Index

3.3.1 Detection of Anti thyroid antibodies TPOAb and TGAb

On the day of death, the rats in each group were anesthetized with 3% sodium pentobarbital (0.1 mL/100 g) intraperitoneally without food or water. Blood was collected from each rat through the abdominal aorta, and 5mL of whole blood was collected. The serum was obtained after centrifugation (3000 r/min) for 10 min.TPOAb and TGAb levels were detected according to the ELISA Kit instructions of TPOAb and TGAb in rats.

3.3.2 Detection of thyroid function and IL-38

Rat serum was obtained by the same method as above, and the concentrations of T3, FT3, T4, FT4, TSH and IL-38 were detected according to the instructions of the ELISA kit for rat T3, FT3, T4,FT4, TSH and IL-38.

3.3.3 Histopathological observation

The thyroid gland of rats was taken and weighed, fixed with 10%neutral formalin solution for 48 hours, then embedded in paraffin,and continuously sectioned for 6 μM thick, stained with hematoxylin and eosin, dehydrated and transparent, and then sealed with neutral gum. The size and morphology of epithelial cells and follicles,the degree of lymphocyte infiltration and the degree of interstitial fibrosis were observed under the electron microscope. The grading index of pathological changes was observed under light microscope:normal thyroid tissue was 0; Focal thyroiditis was 1 point; Severe thyroiditis not exceeding 40% was counted as 2 points; 40% to 80%of diffuse thyroiditis was counted as 3 points; More than 80% of diffuse thyroiditis was counted as 4 points[8].

3.3.4 Detection of Th17 and Treg by Flow Cytometry

The spleen of 5 rats in each group was taken to prepare the spleen cell suspension, which was filtered with a sieve, and the corresponding staining antibody was added. After incubation, it was tested on the computer. Wherein CD4+IL-17A+is used to determine Th17; CD4+CD25+Foxp3+was used to determine Treg.

3.4 Statistical Method

SPSS 25.0 statistical software is used, and the measurement data is expressed by±s. One way ANOVA test is used for the comparison between multiple groups when the normal distribution is met, and non parametric test is used for the comparison between multiple groups when the normal distribution is not met; LSD test was used for those with uniform variance and tamhanet2 test was used for those with uneven variance. The difference was considered statistically significant (P＜0.05).

4. Results

4.1 Effect of Fuzheng Xiaoying Decoction on Anti thyroid antibody in rats with EAT

The levels of TPOAb and TGAb in the model group were significantly higher than those in the blank group(P＜0.05).This indicates that the model is successful. The levels of TPOAb and TGAb in selenium yeast tablet group and Fuzheng Xiaoying Decoction group were significantly lower than those in model group(P＜0.05).However, there was no significant difference in TPOAb and TGAb levels between the selenium yeast tablet group and the Fuzheng Xiaoying Decoction group. As shown in Table 1.

Tab 1 Effect of the treatment group on TPOAb and TGAb levels of EAT model rats(IU/mL, n=10, ±s)

Tab 1 Effect of the treatment group on TPOAb and TGAb levels of EAT model rats(IU/mL, n=10, ±s)

Note: compare with the blank group,*P＜0.05;compare with the model group,△P＜0.05.

Group TPOAb TGAb blank group 11.20±4.13 130.13±26.69 model group 22.24±5.19* 204.32±34.12*selenium yeast tab Fuzheng Xiaoying Delet group 15.98±3.05*△ 162.34±29.84*△coction group 15.67±4.39*△ 173.98±28.17*△F 10.848 9.966 P 0.000 0.000

4.2 Effect of Fuzheng Xiaoying Decoction on thyroid histopathology in rats with EAT

In the blank group, the thyroid follicles were arranged closely and regularly, and light red glia were evenly distributed in the cavity.The epithelial cells were in low columnar shape, and no lymphocyte infiltration was found. There is a thin-walled fibrous tissue interval between the leaflets, and no fibrosis is seen (as shown in Fig.1a).In the model group, the thyroid follicles are arranged in disorder,the glia are uneven, and some of the follicles are damaged. A large number of lymphocytes can be seen to infiltrate. Some epithelial cells fall off into the follicular cavity, and the interlobular space is slightly widened (as shown in Fig.1b), indicating that the modeling is successful. The glial content in the follicles of the selenium yeast group is less than that of the normal group. It can be seen that some of the follicle structures are damaged, some epithelial cells fall off into the follicle cavity, a small number of lymphocytes are distributed, the interlobular septum is widened, and the fibrous tissue is proliferated. Compared with the model group, the lymphocyte infiltration is reduced (as shown in Fig.1c). In the Fuzheng Xiaoying Decoction group, the thyroid follicle morphology is basically regular,the colloid distribution in the cavity is relatively uniform, and scattered lymphocytes can be seen. Compared with the model group,the follicular destruction and lymphocyte infiltration are improved(as shown in Fig.1D). The pathological grade of thyroid tissue of rats in each group was observed. It was found that the pathological grade of the model group was higher than that of the blank group(P＜0.05).Compared with the model group, the pathological grade of Fuzheng Xiaoying Decoction group decreased(P＜0.05).Meanwhile, the pathological grade of Fuzheng Xiaoying Decoction group was lower than that of selenium yeast group(P＜0.05).As shown in Table 2.

Tab 2 Effect of treatment group on thyroid pathological grade in EAT model rats(n=10, ±s)

Tab 2 Effect of treatment group on thyroid pathological grade in EAT model rats(n=10, ±s)

Note: compare with the blank group,*P＜0.05; compare with the model group,△P＜0.05; compare with the selenium yeast tablet group,#P＜0.05.

Group Pathological grading of thyroid gland blank group model group selenium yeast tablet gro heng Xiaoying Decoctionup group0.50±0.55 2.33±0.82*2.33±0.52 1.17±0.98△14.860 Fuz #F P 0.002

4.3 Effect of Fuzheng Xiaoying Decoction on thyroid function of EAT rats

The levels of T3, FT3, T4 and FT4 in the model group were higher than those in the blank group(P＜0.05): There was no significant difference in TSH levels between the two groups. The T4 level of selenium yeast tablet group and Fuzheng Xiaoying Decoction group was significantly lower than that of model group(P＜0.05).However,there was no significant difference in T3, FT3 and FT4 levels among the selenium yeast tablet group, Fuzheng Xiaoying Decoction group with the model group. As shown in Table 3.

4.4 Effect of Fuzheng Xiaoying Decoction on IL-38 and Th17-Treg in rats with EAT

Compared with the blank group, the level of IL-38 decreased,Th17% increased and Th17/Treg ratio increased in the model group(P＜0.05).The level of IL-38 in selenium yeast tablet group and Fuzheng Xiaoying Decoction group was higher than that in model group(P＜0.05). Th17% and Th17/Treg in the two groups of drug intervention were lower than those in the model group, but there was no statistical difference. There was no statistical difference in Treg%among the groups. As shown in Table 4.

Fig 1 HE staining of rat thyroid tissue(200×)

Tab 3 Effect of the treatment group on thyroid function of EAT model rats(n=10, ±s)

Tab 3 Effect of the treatment group on thyroid function of EAT model rats(n=10, ±s)

Note: compare with the blank group,*P＜0.05;compare with the model group,△P＜0.05.

Group T3(ng/mL) T4(ng/mL) FT3(pmol/L) FT4(pmol/L) TSH(mU/L)blank group 5.78±1.46 177.38±72.03 3.64±1.81 20.08±5.90 20.37±2.96 model group 10.18±1.91* 351.98±82.78* 10.75±3.74* 33.79±8.90* 26.99±3.79 selenium yeast tablet group 8.99±2.75* 232.29±104.34△ 9.44±2.99* 28.03±7.73* 24.48±7.28 Fuzheng Xiaoying Decoction group 8.06±1.98* 213.78±81.74△8.45±2.98* 31.70±5.86* 23.20±6.52 F 7.432 7.454 9.988 6.492 7.030 P 0.001 0.001 0.000 0.001 0.071

Tab 4 Effect of the treatment group on IL-38 and Th17/Treg in rats with EAT model (n=5, ±s)

Tab 4 Effect of the treatment group on IL-38 and Th17/Treg in rats with EAT model (n=5, ±s)

Note: compare with the blank group,*P＜0.05; compare with the model group,△P＜0.05.

Group IL-38(pg/mL) Th17(%) Treg(%) Th17/Treg blank group 143.24±13.07 4.7 73.11±15.94* 9.4 115.49±25.10*△ 6.4△0±1.16 13.55±1.48 0.32±0.09 model group 6±2.89* 15.36±0.62 0.63±0.08*selenium yeast tablet group 5±2.84 14.63±2.93 0.56±0.20 Fuzheng Xiaoying Decoction group 127.07±20.936.94±3.11 15.50±2.52 0.56±0.30 F 12.049 2.817 0.913 2.453 P 0.000 0.072 0.457 0.101

5. Discussion

Th17 lymphocytes are mainly involved in inducing the expression of chemokines and inflammatory cytokines, playing a proinflammatory role, thus causing autoimmune diseases [9];Treg cells can inhibit the development of autoimmune diseases by maintaining autoimmune tolerance and controlling the proliferation of self activated CD4+T cells [10]. The function of Th17 cells is enhanced and the function of Treg cells is decreased, which can make B lymphocytes produce a large number of antibodies. The antibody dependent cytotoxicity, the activation of complement system and the cytotoxicity mediated by natural killer cells (NK cells) jointly cause lymphocytes to infiltrate the thyroid, destroy the thyroid follicular structure, and lead to HT [11]. The level of thyroid autoantibodies is related to the symptoms of depression and decreased quality of life in HT patients, but the main purpose of Western medicine treatment is to control the abnormal thyroid function, and the patients still have symptoms after the recovery of thyroid function [12]. In recent years, a large number of clinical studies have found that Traditional Chinese Medicine treatment has a good clinical effect in reducing HT Antithyroid antibody, improving clinical symptoms and delaying the development of the disease [13].Modern doctors mostly treat the disease from the liver, spleen and kidney. The method of dispersing the disease is to remove blood stasis, phlegm and soft and hard. In this experiment, the basic method of strengthening the spleen and strengthening the body and eliminating phlegm and gall is used to intervene eat for the first time some studies have shown that Chinese herbal formulas can regulate the balance of Th17-Treg cells in autoimmune thyroiditis. For example, Yiqi Huatan Huoxue recipe can enhance the immunosuppressive effect of Th1, Th2 and Treg cells on Th17 cells[14],Qili Xiaoying Decoction can regulate IL-23/IL-17 inflammatory axis[15]. In addition, Yanghe Decoction[16]、Xiaoying mixture[17]、Ruanjian Xiaoying granules[18]can regulate Foxp3 and RORγt gene expression by affecting IL-6, IL-8, IL-10,IL-17 and TNF-α.

HT belongs to the category of "gall disease" in traditional Chinese medicine because of its main clinical symptom of goiter. It is said in the Ji Sheng Fang - treatment of gall tumor and the surgical authentic- gall tumor gate that gall tumor is caused by Qi coagulation,blood stasis and phlegm stagnation The research group believes that gall disease is caused by phlegm Qi blood stagnation in front of the neck, mainly due to the dysfunction of the viscera, while the spleen is the source of Qi and blood biochemistry. "Spleen qi deficiency means that the limbs are not used and the five viscera are uneasy". Therefore, the treatment from the spleen is the key. When strengthening the spleen and replenishing qi, it is necessary to treat the root cause "All diseases are caused by phlegm". The pathological factors are mainly phlegm, which condenses on the front and both sides of the neck, so there are goiter and nodules. Stagnating around the body, there are fatigue, shortness of breath, fear of cold and so on. Therefore, resolving phlegm and dispersing nodules is the target of eliminating galls[4]. The previous study of our research group found that Buzhong Yiqi granule has a good effect on improving HT [5, 19],While Fuzheng Xiaoying Decoction is based on Buzhong Yiqi Decoction and Xiaolong pill, which enhances the function of eliminating phlegm, eliminating Ying and dispersing knots on the basis of strengthening the spleen, and has a more prominent effect on improving the clinical symptoms of HT patients such as goiter [20]. This prescription takes Huangqi as the king, dangshen and Baizhu as the ministers, enhances the medicinal power of Huangqi, and plays the function of Buzhong Yiqi. With the help of Xuanshen,Tubeimu and Muli, it is used to remove phlegm, eliminate galls and disperse knots. Chenpi and Banxia are used to regulate qi and regulate stagnation, replenish qi and prevent backflow, and also remove phlegm. Xiangfu and Danggui regulate qi and relieve depression, nourish blood and camp. Xiakucao is used to dissipate phlegm and swelling. Chuanshanlong can promote blood circulation,remove dampness and unblock collaterals. Gancao is an auxiliary medicine, which is used to strengthen the spleen and replenish qi and harmonize various medicines. According to modern pharmacology research, in the composition of Fuzheng Xiaoying Decoction,Huangqi, Baizhu, Dangshen, Chenpi, Xiagucao, Muli, Danggui,Chuanshanlong, Tubeimu, Xuanshen, Gancao and other drugs have immune regulating effects[21, 22]. In this study, the model was established by high iodine drinking water combined with immune injection, and the successful model was determined by serum antibody and thyroid pathology.The results showed that Fuzheng Xiaoying Decoction could reduce the level of anti thyroid antibody(including TPOAb and TGAb) in rats with EAT, and the therapeutic effect was similar to that of selenium yeast tablets.At the same time,the prescription can improve the degree of lymphocyte infiltration and follicular destruction in the thyroid tissue of the rats.Through the analysis of pathological grading results, it is found that its effect is better than that of selenium yeast tablets, indicating that Fuzheng Xiaoying Decoction has a positive effect on HT patients.

In addition, according to the thyroid function test results of the EAT rat model, it was found that the levels of T3, T4, FT3 and FT4 in the EAT rat model were significantly higher than those in the blank group, but there was no statistical difference in the TSH level between the groups. It is in the stage of hyperthyroidism, equivalent to the early stage of HT. Its hyperthyroidism may be caused by the release of thyroid hormone into the blood after the thyroid follicles are infiltrated and destroyed by lymphocytes and plasma cells. Tang Wei et al. also found that FT3 and FT4 in the EAT model group were significantly higher than those in the blank group (P＜0.05),but there was no significant difference in TSH level [23].But according to Yaoting[24]and Yu Lingying's [25]research results, the TSH level in the EAT model group was higher than that in the blank group(P＜0.05),Hou Liping's research results showed that the levels of FT3, FT4 and TSH in the EAT model group were higher than those in the blank group(P＜0.05)[26]. It is also possible that the level of FT3 and FT4 changes in the same direction as that of TSH due to the modeling method of exogenous thyroglobulin immunity. However,the observation time limit of EAT rats in various literatures is 1-3 months, and the long-term changes of thyroid function are not tracked. The specific reasons need further experimental verification.In this study, Fuzheng Xiaoying Decoction can reduce the level of thyroid hormone T4 in model rats, but there is no statistical significance for the reduction of FT3, FT4 and T3. Previous studies have found that clinical patients with hyperthyroidism often show the syndrome of excess of heart and liver fire[27]and deficiency of heart and liver yin [28]. In the past, Fuzheng Xiaoying Decoction, which is mainly used to strengthen the spleen, replenish qi and eliminate galls and disperse knots, was mainly used in the period of normal thyroid function and hypothyroidism. Whether it can improve the thyroid function of patients with clinical HT and hyperthyroidism still needs further clinical research.

Studies have found that IL-38 is often expressed abnormally in chronic inflammatory diseases[29]. Jialu Xu found that IL-38 concentrations in HT patients were decreased compared with healthy controls[30]. This study also found that the level of IL-38 in the model group was lower than that in the blank group(P＜0.05). IL-38 level increased after drug treatment(P＜0.05). IL-38 can exert antiinflammatory effect by binding with potential receptors such as IL-1 receptor 1, IL-36 receptor and IL-1 receptor 10. One of the main functions of IL-38 is to inhibit the induction of Th17 cytokine related responses[31]. Previous literature has shown that there are abnormally high levels of Th17 cells and pro-inflammatory cytokines related to Th17 in thyroid tissue and/or peripheral blood of HT patients[32].This study found that Th17% and Th17/Treg ratio in the model group were higher than those in the blank group, which was consistent with previous studies. The Th17% and Th17/Treg ratio of the selenium yeast and Fuzheng Xiaoying Decoction treatment group were lower than that of the model group,but the difference was not statistically significant. Therefore, Fuzheng Xiaoying Decoction may improve the condition of HT by increasing the level of IL-38,thereby inhibiting Th17 differentiation and inflammatory response induced by related cytokines.

To sum up, the results of this experiment show that Fuzheng Xiaoying Decoction can reduce the level of Anti thyroid antibody,repair and reduce the destruction of thyroid tissue follicles and lymphocyte infiltration in EAT model rats. Its possible mechanism of action is to regulate the level of IL-38 and thus inhibit Th17 related inflammatory response. This experiment provides an experimental basis for the clinical application of this formula to treat HT. In the future, we can further explore the specific action pathway and provide a new target and research direction for the treatment of HT from the spleen.

(Description of author's contribution:Qiao Jiajun is responsible for jointly completing the experiment, writing the paper and data analysis; Zhang Yuyang and Shi Jingping are responsible for jointly completing the experiment and article verification; Xia Zhongyuan is responsible for experimental guidance and paper guidance. The author of the article declares that there is no conflict of interest.)