Written informed consent regarding participation in the present study was obtained from patients. Samples were collected from 12 human samples used in this experiment. The age range was from 31 to 64; There were 5 male patients and 7 female patients. All samples were determined and collected in Peking University Cancer Hospital (Beijing, China) from January 2020 to January 2022. Use of stored human samples was approved by the Ethics Committees of Peking University Cancer Hospital and the Institute and National Institute for Viral Disease Prevention and Control, China CDC (approval no. IVDC2021-12, Beijing, China).

Emodin and dimethyl sulfoxide were purchased from MilliporeSigma. The antibodies included antibodies for p65 (cat. no. sc-8008), phosphorylated (p-)p65 (cat. no. sc-136548), p50 (cat. no. sc-114), p-p50 (cat. no. sc-271908), c-Rel (cat. no. sc-6955), cyclin D1 (cat. no. sc-8396), c-Myc (cat. no. sc-40), PCNA (cat. no. sc-56), TLR4 (cat. no. sc-293072), IRF3 (cat. no. sc-33641), AKT1/2/3 (cat. no. sc-56878), MEK1/2 (cat. no. sc-81504), Bcl-2 (cat. no. sc-7382), Bax (cat. no. sc-7480). The above antibodies are from Santa Cruz Biotechnology, Inc., WB dilution concentration is 1:200, IHC and IFA dilution concentration is 1:50. MyD88 (cat. no. ab113739; Abcam, WB dilution concentration is 1:2,000; IFA dilution concentration is 1:200) and β-actin (cat. no. AB0145-200; OriGene Technologies; 1:5,000), HRP-AffiniPure Goat Anti-Rabbit IgG (H+L) Secondary (cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc.), HRP-AffiniPure Goat Anti-Mouse IgG (H+L) Secondary (cat. no. 115-035-003; Jackson ImmunoResearch Laboratories, Inc.).

PTC cell lines TPC-1 and IHH4 were used (14–16). TPC-1 and IHH4 cell lines were purchased from Zhejiang Meisen Cell Technology Co., Ltd. Cells were identified by short tandem repeat analysis and did not exhibit contamination or infection with mycoplasma, bacteria or fungi. Cells were cultured in DMEM (cat. no. 11965118; Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum (cat. no. 10270106; Thermo Fisher Scientific Inc.) and 1% penicillin-streptomycin (cat. no. 15140122; all Thermo Fisher Scientific Inc.) in an incubator at 37°C with 5% CO₂. Emodin was dissolved in buffer containing 0.1% DMSO. The cells in treatment groups were separately exposed to 20 and 40 µM emodin; cells in the mock group were exposed to buffer containing only 0.1% DMSO. Cells were exposed to treatment at 37°C for 24 h. Considering that DMSO at ≤0.1% has no significant effect in cell activity-related experiments (17), a blank cell control was not included.

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8; cat. no. CK04; Dojindo Molecular Laboratories, Inc.) according to manufacturer's instructions. Briefly, after cells were cultured with emodin (10, 20, 40, 60, 80 or 100 µM) at 37°C for 24 h, 10 µl CCK-8 was added to each well at 37°C and 5% CO₂ for 1 h. The optical density (OD) at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Inc.). Relative cell viability was calculated by comparing OD₄₅₀ of the experimental group to that of the control group. The cell survival rate was calculated as follows: Cell survival rate (%)=(Experimental OD-blank OD)/(control OD-blank OD) ×100%.

Surgically removed tumor tissues were fixed in 10% buffered formalin solution at 4°C for 12 h and paraffin sections of thickness of 2 µm were routinely prepared. The paraffin sections of PTC tissue were dewaxed and dehydrated with xylene and alcohol and repaired using a microwave at 100°C for 30 min. Samples were blocked with 3% hydrogen peroxide (cat. no. AR1108; Wuhan Boster Biological Technology, Ltd.) at room temperature (RT) for 10 min. After blocking with 10% normal goat serum (cat. no. AR1009; Wuhan Boster Biological Technology, Ltd.) at room temperature (RT) for 15 min, sections were incubated with primary antibodies (p65: cat. no. sc-8008; p-p65: cat. no. sc-136548; p50: cat. no. sc-114; p-p50: cat. no. sc-271908) at 4°C overnight, and secondary antibodies (Enzyme-labeled goat anti-mouse IgG polymer: PV-6002, Enzyme-labeled goat anti-rabbit IgG polymer: PV-6002; Beijing Zhong Shan Goldenbridge Biotechnology Company Ltd.) at 37°C for 40 min. For visualization, slices were incubated with 3,3-diaminobenzidine tetrahydrochloride (cat. no. AR1000; Wuhan Boster Biological Technology, Ltd.) at RT for 3 min, counterstained with hematoxylin (cat. no. AR 0005; Wuhan Boster Biological Technology, Ltd.) at RT for 1 min, dehydrated and seal the sample with resin and cover glass (cat. no. 10212450C; Citotest Scientific Co., Ltd.). Then use a light microscope to enlarge it by 400 times. Analysis was performed using ImageJ (version 1.8.0; National Institutes of Health) measurement results.

After washing cells with PBS three times, cultured cells were collected and harvested by centrifugation at 37°C at 500 × g for 10 min. Resulting pellets were lysed at 4°C for 1 h using Mammalian Protein Extraction kit (cat. no. CW0889; CoWin Biosciences) with protease inhibitor Cocktail set III (1%; v/v; cat. no. 535140; Merck KGaA), followed by centrifugation at 4°C at 500 × g for 10 min and supernatants were collected. Protein concentration was estimated using a BCA protein assay kit (cat. no. 71285-3; Merck KGaA). Cell lysates (70–100 µg/lane) were separated by 12% SDS-PAGE and electroblotted onto nylon membranes. The membranes were blocked with Tris-buffered saline (pH 7.6) containing 0.05% Tween-20 and 5% skimmed milk at RT for 1 h, then incubated with antibodies [p65 (cat. no. sc-8008); p-p65 (cat. no. sc-136548); p50 (cat. no. sc-114); p-p50 (cat. no. sc-271908); c-Rel (cat. no. sc-6955); cyclin D1 (cat. no. sc-8396); c-Myc (cat. no. sc-40); PCNA (at. no. sc-56); TLR4 (cat. no. sc-293072); IRF3 (cat. no. sc-33641); AKT1/2/3 (at. no. sc-56878); MEK1/2 (cat. no. sc-81504); Bcl-2 (cat. no. sc-7382); Bax (cat. no. sc-7480). The above antibodies are from Santa Cruz Biotechnology, Inc., WB dilution concentration is 1:200. MyD88 (cat. no. ab113739; Abcam; WB dilution concentration is 1:2,000) and β-actin (cat. no. AB0145-200; OriGene Technologies, Inc.; 1:5,000)] overnight at 4°C. The membranes were incubated with secondary antibodies [rabbit secondary antibody (cat. no. 111-035-003); mouse secondary antibody (cat. no. 115-035-003); The above antibodies are from Jackson ImmunoResearch Laboratories, Inc., WB dilution concentration is 1:5,000] at RT for 1 h. The blots were developed using Western Lightning Plus-ECL (cat. no. NEL105001EA; PerkinElmer, Inc.). Semi-quantitative analysis was performed using a Clinx ChemiCapture System with ChemiScope 6000 (Clinx Science Instruments Co., Ltd.). Using ImageJ (version 1.8.0; National Institutes of Health) measures gray values.

Firstly, 0.4% paraformaldehyde was used to fix cells at RT for 20 min. Following treatment with 0.3% Triton-X100 at RT for 30 min and blocking with 5% BSA for 1 h at RT, cells were incubated with antibodies [p65 (cat. no. sc-8008); p-p65 (cat. no. sc-136548); p50 (cat. no. sc-114); p-p50 (cat. no. sc-271908); c-Rel (cat. no. sc-6955); cyclin D1 (cat. no. sc-8396); c-Myc (cat. no. sc-40); PCNA (cat. no. sc-56); TLR4 (cat. no. sc-293072); IRF3 (cat. no. sc-33641); AKT1/2/3 (cat. no. sc-56878); MEK1/2 (cat. no. sc-81504); Bcl-2 (cat. no. sc-7382); Bax (cat. no. sc-7480). The above antibodies are from Santa Cruz Biotechnology, Inc., IFA concentration is 1:50. MyD88 (cat. no. ab113739; Abcam; 1: 200)] at 4°C overnight. Cells were incubated with secondary antibodies (1:200; Alexa Fluor 488 anti-rabbit or Alexa Fluor 568 anti-mouse; cat. nos. A32731 and A32723, respectively; both Thermo Fisher Scientific, Inc.) at 37°C for 1 h. Following counterstaining with 1 µg/ml DAPI (Beyotime Institute of Biotechnology) at RT for 20 min, images were captured using a Leica TCS SP8 confocal microscope (Leica Microsystems, Ltd.).

Cells were digested with trypsin, a total of 1,000 cells/well was seeded in six-well plates and incubated at 37°C for 12 h. Cells were treated with different concentrations of emodin (20, 40 µM) at 37°C for 7 days. Next, 0.4% paraformaldehyde was used to fix cells at RT for 20 min. And stained with crystal violet at RT for 3 min and images were taken under a light microscope (magnification, ×40 times. When there are more than 50 cells, it can be judged as a colony. Using ImageJ (version 1.8.0; National Institutes of Health), colonies were counted (18).

Wound healing assay was performed to evaluate cell migration. Cells were seeded in a six-well plate and grew to confluence rate of 100%, followed by scratching the monolayer with a 200-µl pipette tip to create a wound. Plates were washed to remove floating cells and debris, DMEM containing 1% fetal bovine serum was added. Then treated with emodin (20, 40 µM) at 37°C for 24 h. The cell migration images were captured at 0 and 24 h post-treatment using a light microscope (magnification, ×40). Each group contained ≥3 independent wells.

Statistical analysis was performed using SPSS (version 22.0; IBM Corp.). Quantification of IFA as integrated option density) was performed using ImageJ (version 1.8.0; National Institutes of Health). All experiments were conducted ≥3 times. All data are presented as the mean ± SD. Difference among cells treated with different concentrations of emodin and mock cells in CCK-8 was determined using unpaired t test. Significant differences between cells treated with different concentrations of emodin and mock cells in western blotting, IHC, IFA, colony formation and wound healing assay were determined using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

An increase in NF-κB activity has been described in numerous types of malignant tumor (19). Tissue sections of PTC at different stages, including three tumor (T)1 node (N)0 metastasis (M)0, three T1N1M0 and three T3N1M0, as well as three benign hyperplastic tissues, were subjected to IHC with antibodies for NF-κB components (Fig. 1A). Compared with benign thyroid hyperplasia tissue, more staining was observed in PTC tissue via IHC analysis of p65 and p50 (Fig. 1B and C), particularly in PTC T1N1M0 and T3N1M0. Quantitative assay of the signal intensity revealed increased levels of p65 and p50 in PTC tissue and the percentage of the positive area increased with progression of tumor stages. Subsequently, IHC of p-p65 and p-p50 demonstrated that more staining were detected in PTC tissues (Fig. 1D and E). Significantly higher percentages of positive areas of p65, p50, p-p65 and p-p50 were calculated for PTC compared with benign tissues in the quantitative assays.

Prior to testing the effect of emodin on the NF-κB components and upstream TLR4 signaling, thyroid cancer cell lines IHH4 and TPC-1 were treated with various concentrations of emodin (10, 20, 40, 60, 80 or 100 µM) to assess cytotoxicity. Cells were harvested at 24 h post-exposure and subjected to CCK-8 assay. The data of the individual preparations were normalized with that of the cells without emodin, set as 100%. As emodin concentration increased, the survival rate of cells decreased. Compared with the preparations treated with DMSO not containing emodin (0 µM), the viability of the cells treated with 20 and 40 µM emodin was slightly decreased, while cells exposed to ≥60 µM emodin had significantly decreased viability (Fig. 2A). Based on the results of CCK-8 assays, 20 and 40 µM emodin were used in subsequent experiments. At 24 h after exposure, emodin-treated cells and the mock cells exposed to 0.1% DMSO buffer were harvested. Western blotting of cellular lysates revealed that the signals of NF-κB components p65, p50 and c-Rel, as well as p-p65 and p-p50, were significantly weaker in cells treated with emodin compared with the mock group, the ratio of p-p65 to p65, p-p50 to p50 were significantly decreased (Fig. 2B). IFA also revealed significantly weaker green staining in emodin-treated IHH4 and TPC-1 cells (Fig. 2C). The effect of 40 µM emodin on various NF-κB components was stronger than that of 20 µM.

The levels of elements in the upstream regulatory pathway for NF-κB, including TLR4, MyD88, IRF3, AKT and MEK, in IHH4 and TPC-1 cells were examined after exposure to emodin or DMSO. Western blotting demonstrated that expression of TLR4, MyD88, IRF3, AKT and MEK in the emodin-treated cells was significantly weaker than in the mock cells exposed to DMSO (Fig. 2D). IFA revealed that the fluorescence intensities of TLR4, MyD88, IRF3, AKT and MEK in the emodin-treated cells was significantly weaker than in the mock cells exposed to DMSO (Fig. 2E) in emodin-treated cells. This indicated that emodin treatment of thyroid cell lines inhibited the upstream regulatory TLR4 signaling pathway of NF-κB.

To examine the effects of emodin on cellular levels of proteins involved in cell proliferation and apoptosis, thyroid cancer cell lines treated with or without emodin were subjected to western blotting and IFA. Compared with the control group, the protein expression levels of PCNA, cyclin D1 and c-Myc in the cells treated with emodin significantly decreased (Fig. 3A). Significantly lower fluorescence intensities in emodin-treated IHH4 and TPC-1 cells were also identified in PCNA, cyclin D1 and c-Myc (Fig. 3B). Furthermore, the levels of apoptotic Bcl-2 and Bax were examined. Both western blotting and IFA revealed a decrease and an increase in Bcl-2 and Bax levels, respectively, in the cells treated with emodin (Fig. 3C and D).

To assess the influence of emodin on tumor characteristics such as proliferation, apoptosis, migration, and invasion of IHH4 and TPC-1 cells (20), cells were assessed using a colony formation assay after treatment with and without emodin for 7 days. A lower number and smaller size of colonies were observed in cells receiving 20 and 40 µM emodin compared with those in the mock cells (Fig. 4A). The wound healing assay showed that the number of TPC-1 cells treated with emodin for 24 h was lower than that of the control group (Fig. 4B). This demonstrated that exposure to emodin decreased colony formation and migration of thyroid cancer cells in vitro.