The experiment complied with the Chinese Act on Animal Experimentation, which implements the Directive 2011/588 on the Protection of Animals used for Scientific Purposes. The animal experiments were approved by the Subcommittee of Experimental Animal Ethics of the Academic Committee of Beijing University of Chinese Medicine, no: BUCM-4-2019070303-3003. Working license number of laboratory animal practitioners: 1117052400087. And all experiments were carried out in compliance with the ARRIVE guidelines.
Fifty female SPF(Specific pathogen free) Lewis rats, aged 4 weeks, weighing 80 ± 10 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing, China; license no: SCXK (Jing) 2016-0006.
Diosgenin (High Performance Liquid Chromatography(HPLC) ≥ 98%, lot: C10J9Q52616. Shanghai Yuanye Bio-Technology Co., Ltd, Shanghai, China.) was dissolved in 0.5% carboxymethyl cellulose sodium salt solution (lot: SL29151601, Coolaber, Beijing, China) and 0.02% dimethyl sulfoxide (lot: 1129E031, Solarbio, Beijing, China) was added to prepare the suspension for subsequent use.
Sodium iodide solution was prepared by dissolving 50 mg of sodium iodide in 100 ml of water to produce a 0,05% solution. It is important to keep this solution away from strong light and to use it immediately following preparation (NaI, CAS no: 7681-82-5, 99% AR, Maya-R).
Preparation of emulsifier: Firstly, a 0.1% porcine thyroglobulin (PTg) solution was prepared with phosphate buffer saline(PBS), and then mixed with complete Freund's adjuvant (CFA) with an equal volume ratio; the mixture was then ground into an emulsion. The final concentration of PTg was 0.05%, i.e. 100 ug PTg/0.2 ml emulsifier. The preparation method of incomplete Freund’s adjuvant (IFA) was the same as for CFA (CFA, lot: SLBW7430, Sigma, St. Louis, MO, USA; IFA, lot: SLBZ0619, Sigma; PTg, lot: 018K7012, Sigma).
AIT induction in rats and drug treatment
Protocol for the AIT animal model 
Ten rats were randomly selected as the normal group, and the rest were modeled. From the first week of the experiment, model rats were free to drink 0.064% NaI, while non-model rats were free to drink double distilled water. In the third week, the model rats in each group were immunized for the first time: Thyroid globulin emulsion with CFA was injected into the foot pad, and subcutaneously into the back and neck of rats at multiple points (0.2 ml per injection site). Two series of injections were given with an interval of 2 days during one week. Intensified immunization from the 4th to 8th week: Injections of 0.2 ml were performed into the foot pad and subcutaneously into the back and neck of rats once a week. To verify the success of model building, blood was collected from the orbital vein, and the levels of TPOAb antibody in peripheral serum was detected by Elisa. Three model rats were randomly selected for observation of pathological changes in the thyroid gland.
Fifty rats were randomly divided into a normal group, an AIT-model group, a diosgenin low-dose group, a diosgenin middle-dose group, and a diosgenin high-dose group according to their weight, with 10 rats in each group. After modeling, forty AIT rats were randomly divided into four groups according to their serum TPOAb value. And the drug was administered. Rats in the diosgenin low-dose group (10 mg/kg.d), the diosgenin middle-dose group (20 mg/kg.d), and the diosgenin high-dose group (40 mg/(kg.d) were given corresponding drug suspensions, and rats in the normal group and model group were given the same volume of deionized water 1 ml/100 g.d. The treatments were continuously administered for 8 weeks.
Serum and pathological examination
After treatments were completed, rats were anesthetized with 1% pentobarbital sodium, and blood was taken from the abdominal aorta to obtain serum. T3, T4, FT3, FT4, TSH, TRAb and TgAb were detected at the Beijing Sino-UK Institute of Biological Technology. The expression levels of TPOAb were detected by enzyme-linked immunosorbent assay (TPOAb ELISA Kit, lot: C0336030355, Cusabio, Wuhan, China). Unilateral thyroid lobes were taken and fixed in 4% paraformaldehyde for pathological observation. The contralateral thyroid lobe was immediately placed into liquid nitrogen for transcriptome and PCR detection.
The diosgenin high-dose group was detected by transcriptome(n=5), RT-PCR(n=5) and IHC-P(n=3), excluding diosgenin low-dose and middle-dose group.
Transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd., Shanghai, China.
RNA extraction and library preparation
Following the collection of unilateral thyroid lobes from rats, they were quickly placed in liquid nitrogen. Total thyroid lobes RNA was extracted using a mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) following the manufacturer’s protocol. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with an RNA Integrity Number (RIN) ≥7 were subjected to subsequent analysis. The libraries were constructed using a TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. These libraries were then sequenced on an Illumina sequencing platform (Illumina HiSeq X Ten) and 125/150 bp paired-end reads were generated.
Quality control and mapping
Raw data (raw reads) were processed using Trimmomatic Reads containing ploy-N and low-quality reads were removed to obtain clean reads. Subsequently the clean reads were mapped to the reference genome using HISAT2
Gene-level quantification, analysis of differentially expressed genes (DEGs), cluster analysis, Gene Ontology (GO), and KEGG enrichment
The fragments per kilobase of transcript per million mapped reads (FPKM) value of each gene was calculated using the Cufflinks package, and the read counts of each gene were obtained using HTSeq-count. DEGs were identified using the DESeq R package functions estimateSizeFactors and nbinomTest. A P-value <0.05 and foldChange >2 or foldChange <0.5 were set as thresholds for significantly different expression. Hierarchical cluster analysis of DEGs was performed to explore gene expression patterns. GO enrichment and KEGG pathway enrichment analysis of DEGs were performed, respectively, using R based on hypergeometric distribution.
Transcript-level quantification, analysis of differentially expressed transcript , cluster analysis, GO, and KEGG enrichment
For transcript-level quantification, FPKM and read count values of each transcript (protein_coding) were calculated using Bowtie 2 and eXpress. DEGs were identified using the DESeq functions estimateSizeFactors and nbinomTest. A P-value <0.05 and foldChange >2 or foldChange <0.5 were set as the thresholds for significantly different expression. Hierarchical cluster analysis of DEGs was performed to explore transcript expression patterns. GO enrichment and KEGG pathway enrichment analysis of DEGs were performed, respectively, using R based on the hypergeometric distribution.
Real-time quantitative RT-PCR
RNA extraction and real-time quantitative RT-PCR were conducted by OE biotech Co., Ltd., Shanghai, China.
Quantification was performed using a two-step reaction process: reverse transcription (RT) and PCR. Each RT reaction consisted of 0.5 μg RNA, 2 μl of 5×TransScript All-in-One SuperMix for qPCR and 0.5 μl of gDNA Remover, in a total volume of 10 μl. Reactions were performed in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) for 15 min at 42 ℃, and subsequently for 5 s at 85 ℃. The 10 μl RT reaction mix was then diluted ×10 in nuclease-free water and held at -20 ℃.
Real-time PCR was performed using a LightCycler 480 Ⅱ Real-time PCR Instrument (Roche, Basel, Switzerland) with a 10 μl PCR reaction mixture that included 1 μl of cDNA, 5 μl of 2×PerfectStartTM Green qPCR SuperMix, 0.2 μl of forward primer, 0.2 μl of reverse primer, and 3.6 μl of nuclease-free water. Reactions were incubated in a 384-well optical plate (Roche) at 94 ℃ for 30 s, followed by 45 cycles at 94 ℃ for 5 s, and then 60 ℃ for 30 s. Each sample was run in triplicate for analysis. At the end of the PCR cycles, a melting curve analysis was performed to validate the speciﬁc generation of the expected PCR product. The primer sequences were designed in the laboratory and synthesized by TsingKe Biotech(Beijing, China) based on the mRNA sequences obtained from the National Center for Biotechnology Information(NCBI) database as table 5. The expression levels of mRNAs were normalized to β-actin and were calculated using the 2-ΔΔCt method.
The main steps of immunohistochemical detection were as follows: dewaxing, hydration, 3% hydrogen peroxide treatment, high temperature antigen repair, goat serum sealing, primary antibody at 4 ℃ overnight, rewarming with PBS washing, dropping secondary antibody, diaminobenzene color rendering, hematoxylin staining, hydrochloric acid and alcohol differentiation, tap water washing, dehydration, and sealing. Photomicrographs were taken under a microscope using an Image-Pro Plus 6.0, Media Cybernetics system to detect absorbance, analyze the integral optical density (IOD/Area) of cAMP (lot: ab76238, abcam, Cambridge, MA, USA), PKA (alpha/beta/gamma, phosphor T197, ab75991, abcam, USA), and Creb (phosphor S133, ab32096, abcam, USA), and to carry out statistical processing.
SPSS v20.0 statistical software was used to process the data which were expressed using the mean and standard deviation (x ± sd). The data accords with normal distribution and the variance is homogeneous, which is tested by one-way ANOVA, and LSD method is used for comparison between groups. If the data does not conform to normal distribution or variance is uneven, nonparametric test is used. And appropriate correction for multiple comparisons. A difference was regarded to be statistically significant at P < 0.05.