The effects of adipose-derived mesenchymal stem cells combined with sodium selenite on Hashimoto’s thyroiditis

Abstract

Recent research found that sodium selenite (Na₂SeO₃) could ameliorate oxidative damage in patients with Hashimoto’s thyroiditis (HT). Additionally, the effects of adipose-derived mesenchymal stem cells (AMSCs) in an animal model of HT were also reported. However, the effects of AMSCs combined with Na₂SeO₃ on HT are unknown. We investigated the combined effects of AMSCs and Na₂SeO₃ in a rat model of HT and the in vitro effect of Na₂SeO₃ on AMSCs using gene microarray analyses. In the HT rat model, the combination of AMSCs and Na₂SeO₃ restored thyroid tissue structure to that of normal controls and increased the levels of most antioxidant and inflammatory cytokines examined, but decreased the levels of interleukin 10 (IL-10) in HT thyroid tissues. At 0.5-20 µM, Na₂SeO₃ promoted AMSC growth and increased the levels of reduced glutathione and total antioxidant capacity in AMSCs (P<0.05). Na₂SeO₃ increased the levels of hepatocyte growth factor (HGF), transforming growth factor beta (TGF-β), and stem cell factor (SCF) in AMSC culture supernatants. The results of the gene microarray analyses showed that the expression levels of certain genes involved in mitosis, DNA replication and repair, ubiquitination, synthesis and metabolism, and mitochondrial transport changed in response to Na₂SeO₃ treatment. In conclusion, the combination of AMSCs and Na₂SeO₃ restored the function and structure of the thyroid in an HT model, and Na₂SeO₃ promoted the growth, improved the secretion, and the antioxidant capacity of AMSCs in vitro. This combination treatment may provide a new therapy for patients with HT.

Introduction

Hashimoto’s thyroiditis (HT) is a common autoimmune thyroid disease and is especially prevalent in coastal areas [1]. The cause of HT involves many factors, including genetic susceptibility, inflammation, excess iodine intake, and infection [2]. Presently, treatment for HT is limited to antithyroid agents or hormone replacement therapy for hypothyroidism. As the pathogenesis of HT is unclear, its etiology needs to be further explored.

Consumption of excess iodine is a high-risk environmental factor in HT development, but the mechanism by which excess dietary iodine induces autoimmune thyroiditis is still unclear [3]. Previous reports indicated that excess iodine intake might lead to thyroid tissue injury by inducing immune cell infiltration into the thyroid [4], causing oxidative stress and eventually triggering apoptosis-signaling pathways [5]. Decreased autophagy and enhanced apoptosis were observed in the thyroid tissues of patients with HT [5].

Selenium is an essential nutrient for human health and plays an important role in human thyroid physiology [6]. Selenium-dependent enzymes, such as glutathione peroxidase, act as antioxidants and modify redox status in the thyroid. The lack of glutathione peroxidase activity might contribute to oxidative damage in thyroid cells and the initiation of thyroid autoimmunity [7]. In rats, selenium supplementation was shown to prevent oxidative damage in the thyroid [8]. A recently published clinical trial reported that selenium compounds played significant roles in L-thyroxine therapy by serving as adjuvants and reducing the serum levels of antithyroid peroxidase antibodies in patients with autoimmune thyroiditis [9].

Adipose-derived mesenchymal stem cells (AMSCs) have low immunogenicity in vivo and a high capacity for rapid expansion in vitro. Previous research found that MSCs could migrate into areas of inflammation, release high levels of chemokines, and interact directly with immune cells to regulate abnormal immunity [10]. The administration of AMSCs (both syngeneic and allogeneic) to mice with autoimmune thyroiditis has been shown to suppress T-lymphocyte infiltration into the thyroid [11]. However, the therapeutic roles and specific mechanisms by which these cells function still need to be clarified.

There are no reports on the effects of AMSCs combined with sodium selenite (Na₂SeO₃) on autoimmune thyroiditis. In the present study, we investigated the effects of AMSCs combined with Na₂SeO₃ in a rat model of HT and further explored the effects and mechanisms of Na₂SeO₃ on AMSCs in vitro.

Materials and methods

Preparation of AMSCs

This study was approved by the Institutional Animal Ethical Committee, Qingdao, and the Ethics Committee of the Affiliated Hospital of Qingdao University. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Inguinal fat pads were excised from 8-week-old female Wistar rats for the preparation of AMSCs. Briefly, the fat tissue was cut into pieces and digested with 0.05% collagenase type I (Sigma-Aldrich, St. Louis, MO, USA) in 5% bovine serum albumin (Sigma-Aldrich), and subsequently filtered through a 250-μm nylon mesh and centrifuged. The cell pellet was resuspended in AMSC medium containing DMEM (Gibco, Waltham, MA, USA) and 10% fetal bovine serum (Gibco) and cultured at 37°C in 5% CO₂ and 95% O₂.

AMSCs were passaged 3 times prior to harvest. Cell surface marker analysis was performed using flow cytometry for CD31, CD45, CD73, CD44, CD105, and CD90 (Chemicon, Temecula, CA, USA). The specific fluorescence of 10,000 cells was analyzed with a FACScalibur instrument (Becton Dickinson, Franklin Lakes, NJ, USA) using Cell Quest Pro software. Adipogenic and osteogenic differentiation markers were analyzed, as previously described [12,13]. Calcium deposition was determined using von Kossa staining (Gibco), and intracellular droplets of adipocytes were visualized using oil red O staining (Sigma).

Experimental rat model of autoimmune thyroiditis

A total of 70 female Wistar rats (6 weeks of age), weighing 80-100 g, were purchased from the Nanjing Laboratory Animal Center (Nanjing, China). Rats were fed specific pathogen-free mouse chow and water ad libitum and housed at a temperature of 22-23°C and a humidity of 60% with a 12-h light/dark cycle.

Experimental autoimmune thyroiditis (EAT) was induced by the injection of porcine thyroglobulin (Tg, Sigma-Aldrich) and maintained with high iodine water (500 mg/L) feeding. For induction, 50 mg of porcine Tg in deionized water (0.5 mL) was emulsified in Freund’s complete adjuvant (1:1, v/v, Sigma-Aldrich), and 1 mL was administered subcutaneously (s.c.) to each rat. The booster injections on days 7, 21, and 28 were given s.c. using 50 mg porcine Tg in incomplete Freund’s adjuvant (Sigma-Aldrich), using the same dosage and injection method. The success of the EAT model was confirmed by examining the serum levels of FT3, FT4, TSH, Thyroid peroxidase antibody (TPO Ab), and Thyroglobulin antibody (Tg Ab), and images of the thyroid gland.

Fifty rats were injected with porcine Tg, which successfully induced EAT in 90% of the animals. Forty-four EAT rats were randomly divided into 4 groups: HT control group (n=10), AMSC group (n=12), Na₂SeO₃ group (n=10), and AMSC + Na₂SeO₃ group (n=12). Rats fed normal water and chow throughout the experiments were considered normal controls (n=10). Rats in the HT control group were fed high iodine water (500 mg/L sodium iodide). Rats in the AMSC group were treated with 4 AMSC infusions via the vena caudalis from the third day after diagnosis of EAT (1 × 10⁶ AMSCs per rat, one injection each week) for 4 weeks. Rats in the Na₂SeO₃ group were treated with 0.2 mg Na₂SeO₃/kg/day (S5261, Sigma-Aldrich) by intragastric administration from the third day after diagnosis of EAT for 35 days. Rats in the AMSC + Na₂SeO₃ group were treated with the AMSC infusions as described above and 0.2 mg Na₂SeO₃/kg/day for 35 days. All rats were sacrificed on day 63 for analysis.

Determination of serum selenium levels and thyroid function

Serum selenium concentrations were measured in duplicate using inductively coupled plasma mass spectrometry (ICP-MS, Perkin-Elmer SCIEX ELAN 6000, Waltham, MA, USA). A calibration curve was established for 1-400 µg selenium/L (r²>0.99). The limit of detection was 0.5 µg/L. Serum levels of FT3, FT4, TSH, TPO Ab, and Tg Ab were examined, according to the manufacturer’s protocol (Roche Diagnostics GmbH, Mannheim, Germany). All tests were repeated 3 times.

Pathological examination and AMSC homing assay

Thyroid samples were fixed in 10% neutralized formalin, embedded in paraffin, and cut into 4-μm sections. Tissue sections were counterstained with hematoxylin and observed using optical microscopy. The homing efficiency of injected AMSCs was assayed by labeling AMSCs with the red fluorescent dye CM-Dil (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Animals were sacrificed 2 days after injection, and the presence of CM-Dil-labeled cells was examined in thyroid tissue using fluorescence microscopy.

Cytokine analysis

Supernatant samples of the thyroid tissue extract were assayed to determine levels of cytokines in T helper type 1 (Th1) and Th2 cells using a multiplex cytokine/chemokine kit for tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), hepatocyte growth factor (HGF), stem cell factor (SCF), transforming growth factor beta (TGF-β), and interleukins (IL-2, IL-4, IL-6, and IL-10), according to the manufacturer’s instructions (Millipore, Bedford, MA, USA). To determine cytokines released by AMSCs, cell culture supernatants were used. Levels of vascular endothelial growth factor (VEGF), TGF-β, Indoleamine 2,3-dioxygenase (IDO), SCF, and HGF were measured using enzyme-linked immunosorbent assays, according to the manufacturer’s instructions.

AMSCs cultured with sodium selenite

AMSCs (at passage 4) at a density of 10,000 cells/well were seeded onto 6-well cell culture plates and treated with different concentrations (0.1-50 µM) of Na₂SeO₃ (Sigma-Aldrich), as previously described [14]. Untreated cells were considered the control group. AMSCs (2 × 10⁵) were plated into 60-mm dishes one day prior to exposure to Na₂SeO₃. The number of AMSCs was counted each day using a Coulter counter (Beckman Coulter, Brea, CA, USA) and photographed under microscopy after 4 days (Nikon, Tokyo, Japan).

Gene microarray analysis

Microarray analysis was carried out, as previously described [15]. Briefly, RNA was extracted using an RNeasy Mini kit (Qiagen, Venlo, Netherlands), according to the manufacturer’s protocol. Array analysis was carried out using cRNA prepared from equal amounts of RNA (1 μg) pooled from 3 replicates per group. Biotin-labeled cRNA was fragmented at 94°C for 35 min, hybridized overnight onto an Affymetrix mouse 430A 2.0 GeneChip® (Affymetrix, Santa Clara, CA, USA), and scanned using an Agilent Gene Array scanner (Agilent Technologies, Santa Clara, CA, USA). Grid alignment and raw data generation were performed using Affymetrix GeneChip Operating software, version 1.1. A noise value, based on the variance of low-intensity probe cells, was used to calculate the minimum threshold for each GeneChip. Samples were averaged, and the data refined by eliminating genes with signal intensities less than 300 in both groups. Heat maps were generated from greater than or equal to 3-fold changes in gene expression normalized to control tissue using an unsupervised hierarchical cluster analysis.

Quantitative RT-PCR

Total RNA was extracted using the Trizol reagent (Life Technologies), and 1 μg of total RNA was reverse transcribed to generate cDNA using a cDNA synthesis kit (TransGene, Beijing, China). Real-time qPCR analysis was performed in triplicate, with GAPDH as the internal control, using the SYBR Green qPCR SuperMix and StepOnePlus Real-Time Detection System (Applied Biosystems, Waltham, MA, USA), following the manufacturer’s instructions. PCR primers were designed using Primer 3, and their specificity was verified using BLAST (NCBI, Maryland, USA).

Determination of cellular antioxidant status and oxidative damage

Cellular antioxidant status was evaluated using the levels of total antioxidant capacity (T-AOC) and reduced glutathione (GSH). Levels of T-AOC were measured using a previously described colorimetric method [16]. Intracellular GSH concentrations were measured using a commercial assay kit (GSH assay kit, Beyotime). Absorbance was measured at 412 nm, and the data were normalized to cellular protein levels.

Statistical analysis

All statistical analyses were conducted using SPSS, version 17.0 (SPSS Inc., Chicago, IL, USA). All results were expressed as the mean and standard error of the mean. Group data were compared using the Student’s t test or one-way analysis of variance, followed by Tukey’s post hoc test. Differences with a confidence level of 95% or higher were considered statistically significant (P<0.05).

Results

Characterization of AMSCs and animals

AMSCs isolated from rat inguinal adipose tissue were adhered to the plastic surfaces of culture flasks and had a spindle-shaped morphology (Figure 1). After 3 passages, the cells were highly pure and expressed CD₄₄ ⁺ (75.34%), CD₁₀₅ ⁺ (79.26%), CD₉₀ ⁺ (85.77%), CD₇₃ ⁺ (89.63%), CD₃₄ ^- (0.34%), and CD₄₅ ^- (0.14%). The adipogenic and osteogenic differentiations are shown in Figure 1.

Figure 1.

AMSCs and multilineage differentiations. A: AMSCs at passage 4; B: Flow cytometry analysis of AMSCs; C: AMSCs with adipogenic differen-tiation; D: Result of oil red O staining; E: AMSCs with osteogenic differentiation; F: Result of von Kossa staining.

AMSCs combined with Na₂SeO₃ restore thyroid function and structure in HT rats

A rat model of HT was successfully established via injections of porcine Tg and maintained with high iodine water (500 mg/L) feeding. As shown in Table 1, serum levels of FT3, FT4, Tg Ab, and TPO Ab increased, whereas serum levels of TSH decreased in EAT rats compared with normal rats at 10 weeks (P<0.05). After treatment, levels of FT3, FT4, Tg Ab, and TPO Ab in the AMSC and AMSC + Na₂SeO₃ groups significantly decreased, while in these same groups, the levels of TSH increased compared with the HT control group (P<0.05). The levels of FT3, FT4, and TPO Ab in the AMSC + Na₂SeO₃ group were significantly lower than in the AMSC and Na₂SeO₃ groups (P<0.01). Levels of selenium in the AMSC + Na₂SeO₃ and Na₂SeO₃ groups were higher than in the other 3 groups (P<0.05).

Table 1.

Serum levels of FT3, FT4, TSH, TG-ab, TPO-ab and selenium of rats in five groups after treatment

	Control group	HT control group	AMSCs group	AMSCs+Na2SeO3 group	Na2SeO3 group
FT3 (pmol/L)	4.93±1.56	46.31±20.68*	28.37±15.24#	23.46±14.21$,^	34.85±18.97
FT4 (pmol/L)	16.12±8.35	98.32±52.46*	56.32±30.54#	40.97±25.14$,^	83.54±45.87
TSH (mIU/L)	0.01±0.93	<0.001*	0.001±0.054#	0.001±0.061$	<0.001
TG-ab (IU/mL)	16.89±9.35	79.95±43.65*	53.78±27.51#	48.93±26.47$	63.45±38.41
TPO-ab (IU/mL)	10.61±6.21	82.64±45.31*	53.21±25.14#	40.49±22.41$,^	60.83±34.17Δ
Selenium (µg/mL)	0.29±0.17	0.22±0.15	0.26 ± 0.14	0.33 ±0.19$	0.32±0.17Δ

^*P<0.05, compared with Control group;

^#P<0.05, compared with HT control group;

^$P<0.01, compared with HT control group;

^ΔP<0.05, compared with HT control group;

^{^}P<0.01, compared with AMSCs group and Na₂SeO₃ group.

As shown in Figure 2, compared with the normal control group, the follicular structure of the thyroid in the HT control group exhibited moderate to severe damage with obvious infiltration of lymphocytes. In the Na₂SeO₃ group, the structure of the thyroid was partially restored with little lymphocyte infiltration. In the AMSC group, the structure of the thyroid was almost completely restored with little lymphocyte infiltration and additional blood vessels observed around the lobules. However, in the AMSC + Na₂SeO₃ group, the structure of the thyroid was restored to that seen in normal controls after treatment. AMSCs labeled with CM-Dil were observed in the thyroid using fluorescence microscopy, indicating that AMSCs injected into the venous circulation were translocated to the thyroid.

Figure 2.

Pathological results of thyroid tissues. A: Pathological results of thyroid follicular structure in normal control group. B: The follicular structure of the thyroid in the HT control group exhibited moderate to severe damage with obvious infiltration of lymphocytes. C: In the AMSC group, the structure of the thyroid was almost completely restored with little lymphocyte infiltration and additional blood vessels observed around the lobules. D: In the AMSC + Na₂SeO₃ group, the structure of the thyroid was restored to that seen in normal controls after treatment. E: In the Na₂SeO₃ group, the structure of the thyroid was partially restored with little lymphocyte infiltration. F: AMSCs labeled with CM-Dil were observed in the thyroid using fluorescence microscopy.

AMSCs combined with Na₂SeO₃ improve the antioxidant and immunoregulatory capacities of HT rats

We further examined the levels of antioxidant and inflammatory cytokines released by AMSCs in thyroid tissues. Levels of VEGF, TGF-β, SCF, and IDO were decreased in the HT control group compared to the normal control group (P<0.05); after treatment, the levels of VEGF, TGF-β, and SCF in the AMSC and AMSC + Na₂SeO₃ groups increased significantly (P<0.05, Figure 3). Levels of these cytokines were higher in the AMSC + Na₂SeO₃ group compared to the AMSC group (P<0.01).

Figure 3.

Cytokines analysis in thyroid tissues. Levels of VEGF, TGF-β, SCF, IDO and GSH in HT control group decreased compared to the normal control group. After treatment, levels of these cytokines increased in the AMSC + Na₂SeO₃ group compared to the other three groups. *P<0.01, #P<0.05, $P< 0.05, ΔP<0.05 and ^P<0.01. The regulation of immune cytokine levels in AMSC + Na₂SeO₃ group, including TNF-α, IL-2, IFN-γ, IL-4, IL-6 and IL-10, appeared better than in the AMSC and Na₂SeO₃ groups. *P<0.01, #P<0.05, $P<0.05, ΔP<0.05 and ^P<0.01.

The levels of GSH were lower in the HT control group than in the normal control group (P<0.05); after treatment, these levels were higher in the AMSC, Na₂SeO₃, and AMSC + Na₂SeO₃ groups than in the HT control group (P<0.05), especially in the AMSC + Na₂SeO₃ group (P<0.01, Figure 3). The levels of GSH were significantly higher in the AMSC + Na₂SeO₃ group compared to the AMSC and Na₂SeO₃ groups (P<0.01), indicating that the antioxidant ability of HT rats was significantly improved in the AMSC + Na₂SeO₃ group after treatment compared to the AMSC and Na₂SeO₃ groups.

The levels of TNF-α, IL-2, IFN-γ, IL-4, and IL-6 were increased in the HT control group compared to the normal control group (P<0.01); after treatment, these levels were lower in the AMSC, Na₂SeO₃, and AMSC + Na₂SeO₃ groups than in the HT control group (P<0.05), especially in the AMSC + Na₂SeO₃ group (Figure 3). While IL-10 levels in the HT control group were lower than those in the normal control group (P<0.01), after treatment, IL-10 levels were higher in the AMSC and AMSC + Na₂SeO₃ groups than in the HT control and Na₂SeO₃ groups (P<0.05), especially in the AMSC + Na₂SeO₃ group. The regulation of immune cytokine levels in the AMSC + Na₂SeO₃ group appeared better than in the AMSC and Na₂SeO₃ groups (P<0.01).

Sodium selenite promotes AMSC growth

The combination of AMSCs and Na₂SeO₃ achieved better recovery of thyroid and antioxidant and immunoregulatory capacities on HT than treatment with either AMSCs or Na₂SeO₃ alone. To investigate the effects of Na₂SeO₃ on AMSCs, we added different concentrations of Na₂SeO₃ to the culture medium. As shown in Figure 4, Na₂SeO₃ accelerated AMSC growth (at passage 4) at concentrations from 0.5 µM to 20 µM, compared to control AMSCs (P<0.05), especially at 5 µM, whereas 50 µM Na₂SeO₃ inhibited AMSC growth (P<0.01). We selected concentrations of 0.5 µM and 5 µM Na₂SeO₃ to perform subsequent experiments. AMSCs at passages 12-14 that were cultured with Na₂SeO₃ were still more vigorous and grew more rapidly than control AMSCs.

Figure 4.

Sodium selenite promotes AMSC growth. Na₂SeO₃ accelerated AMSC growth (at passage 4) at concentrations from 0.5 µmol/L to 20 µmol/L, compared to control AMSCs. *P<0.05, ΔP<0.01. A: Control AMSCs at passage 4; B: AMSCs cultured with Na₂SeO₃ 0.5 µmol/L; C: AMSCs cultured with Na₂SeO₃ 5 µmol/L.

Sodium selenite repairs oxidative damage and enhances cytokine secretion from AMSCs

Cells can undergo oxidative damage after culture in vitro for long periods of time, so we examined the levels of GSH and T-AOC to evaluate the extent of oxidative damage in AMSCs. The levels of GSH and T-AOC decreased with length of time in culture in vitro (P<0.001), whereas Na₂SeO₃ (both 0.5 µM and 5 µM) increased the levels of GSH and T-AOC in AMSCs (P<0.05, Figure 5).

Figure 5.

Sodium selenite repairs oxidative damage in AMSCs. The levels of GSH and T-AOC decreased with length of time in AMSCs culture in vitro, Na₂SeO₃ (both 0.5 µmol/L and 5 µmol/L) increased levels of GSH and T-AOC in AMSCs. *P<0.001, #P<0.05, $P<0.05, &P<0.05, ΔP<0.05.

The levels of secreted HGF, SCF, and TGF-β decreased in the cell supernatant (comparison of passages 4, 5, and 10, Figure 6) after culture in vitro (P<0.05), whereas Na₂SeO₃ caused an increase in these levels in the AMSC supernatant (P<0.05, data from cells in passage 10 are shown in Figure 6). These findings indicate that Na₂SeO₃ enhances the secretion of cytokines from AMSCs.

Figure 6.

The effect and mechanisms of sodium selenite on AMSCs cytokine secretion. A: The levels of secreted HGF, SCF and TGF-β decreased in the cell supernatant (comparison of passage 4, 5 and 10) after culture in vitro, Na₂SeO₃ caused an increase in these levels in the AMSCs supernatant in passage 10. *P<0.05, #P<0.05, $P<0.05, &P<0.05, ΔP<0.05. B: Microarray analysis of AMSCs cultured with medium including 5 µmol/L Na₂SeO₃ in vitro for 60 days.

Gene microarray analysis

AMSCs were cultured in 5 µM Na₂SeO₃ for 60 days prior to gene microarray analysis to investigate the differences in gene expression between AMSCs cultured in Na₂SeO₃ and control AMSCs. As shown in Figure 6 and Table 2, we found expression levels of 15 genes increased, whereas the expression levels of 14 genes decreased significantly. The expression differences in these genes indicate that Na₂SeO₃ is involved in mitosis, DNA replication and repair, synthesis and metabolism, ubiquitination, mitochondrial transport, cell growth, and migration in AMSCs. We further testified the results of microarray analysis by quantitative RT-PCR. There was a good correlation in the fold change in gene expression levels between the quantitative RT-PCR and microarray data (Table 3).

Table 2.

The genes with changed expression in AMSCs cultured with Na₂SeO₃

Gene symbol	Na2SeO3/Ctl	Gene name
Ubiquitination
Rlim	13.71	ring finger protein, LIM domain interacting
Rmnd5b	-6.08	required for meiotic nuclear division 5 homolog B
Synthesis and metablism
Rasl10b	9	ras-like protein family member 10B
Ppcdc	4.75	phosphopantothenoylcysteine decarboxylase
Hprt1	4.22	hypoxanthine-guanine phosphoribosyltransferase
Hnrnph2	3.4	heterogeneous nuclear ribonucleoprotein A/B
Tyw1	-36.56	S-adenosyl-L-methionine-dependent tRNA 4-demethylwyosine synthase
Iars	-7.37	isoleucine--tRNA ligase, cytoplasmic-like
Cyp19a1	-5.67	cytochrome P450, family 19, subfamily a, polypeptide 1
Atp2b4	-4.21	ATPase plasma membrane Ca2 + transporting 4
Rps25	-3.78	40S ribosomal protein S25-like
Mitochondrial transport
Selenbp1	8.8	Selenium binding protein 1
Ndufa1	8.37	NADH:ubiquinone oxidoreductase subunit A1
Mfsd14a	5.83	major facilitator superfamily domain containing 14A
Timm17b	-31.68	mitochondrial import inner membrane translocase subunit Tim17-B
Mitosis
Ankrd12	8.71	ankyrin repeat domain-containing protein 12
Hira	5.74	protein HIRA-like
Fgd1	5.61	FYVE, RhoGEF and PH domain containing 1
Wbp1	4.98	WW domain binding protein 1
Cetn3	-6.2	centrin-3-like
Cell growth and migration
Zbed6	14.67	PHD finger protein 6
Ppfia3	-6.33	PTPRF interacting protein alpha 3
Dgcr2/idd	-5.92	integral membrane protein DGCR2/IDD
Structural protein
Chid1	9.2	chitinase domain containing 1
Ankra2	-9.38	ankyrin repeat family A member 2
Replication and repair
Alkbh6	5.1	alkB homolog 6
Mmp12	-5.31	matrix metallopeptidase 12
Rsl1d1l1	-5.2	ribosomal L1 domain containing 1-like 1
Tnks2	-4.51	tankyrase 2

Table 3.

Comparison of gene expression levels between microarray and quantitative RT-PCR in AMSCs cultured with Na₂SeO₃

Gene name	Fold change
Rlim	12.98/13.71
Rmnd5b	-6.75/-6.08
Rasl10b	9.58/9
Tyw1	-35.87/-36.56
Selenbp1	9.12/8.8
Timm17b	-31.21/-31.68
Ankrd12	8.13/8.71
Cetn3	-6.52/-6.2
Zbed6	14.1/14.67
Ppfia3	-6.54/-6.33
Chid1	9.87/9.2
Ankra2	-9.81/-9.38
Alkbh6	5.67/5.1

Data are expressed as log₂ gene expression fold change from quantitative RT-PCR/M-value (log₂ gene expression fold change) from microarray.

Discussion

Recent research found that Na₂SeO₃ could ameliorate oxidative damage and decrease the serum levels of TPO Ab in patients with HT [17]. While the effects of AMSCs in an animal model of HT were also reported [11], the effects of AMSCs combined with Na₂SeO₃ on HT were unclear. In the present research, we investigated the effects of AMSCs combined with Na₂SeO₃ in a rat model of HT.

AMSCs combined with Na₂SeO₃ resulted in greater recovery of thyroid structure and function than treatment with AMSCs or Na₂SeO₃ alone. AMSCs are known to home to injured tissues [18]. The presence of CM-Dil in the thyroid indicated that AMSCs injected into the tail vein could be translocated to thyroid tissue, but we didn’t find any thyroid cells containing CM-Dil, suggesting that AMSCs play a role in HT by secreting cytokines but not by transdifferentiating into thyroid cells, consistent with results in a previous report [19].

AMSCs secrete growth factors that modulate the immune system [20,21]. TGF-β, VEGF, and SCF are multifunctional cytokines with anti-inflammatory and regenerative functions [22-24]. IDO has emerged as a key regulator of innate and adaptive immune responses in autoimmune diseases [25]. Increased levels of VEGF, TGF-β, SCF, and IDO in thyroid tissues appear to improve immune system function and promote tissue regeneration. These results are in accordance with a previous EAT study using human AMSCs [26]. AMSCs also can secrete cytokines to effectively regulate immune function [27]. After treatment, levels of TNF-α, IL-2, IFN-γ, IL-4, and IL-6 decreased, and levels of IL-10 increased in the AMSC and AMSC + Na₂SeO₃ groups. Treatment with AMSCs may ameliorate some autoimmune diseases by modulating IL-4 and IFN-γ levels in effector T-cells [27,28]. Moreover, the effects of AMSCs combined with Na₂SeO₃ were greater than with AMSCs alone.

GSH is one of the most important intracellular antioxidants that is released in response to various noxious stimuli, including oxidative stress [29]. Levels of GSH in the Na₂SeO₃ and AMSCs + Na₂SeO₃ groups increased after treatment, and the increase was greater in the AMSCs + Na₂SeO₃ group. Selenite plays a vital role in maintaining the activity of antioxidant enzymes, which reduce levels of hydrogen peroxide and lipid and phospholipid peroxides, thereby blocking the formation of free radicals and reactive oxygen species; such antioxidant activity has proven beneficial in cases of HT [30,31]. These results reveal that the combination of AMSCs and Na₂SeO₃ significantly improve antioxidant activity in rats with HT.

Based on the above findings, we further explored the role of Na₂SeO₃ on AMSCs in vitro and found that it promoted cell growth and maintained the viability of AMSCs. In cultured AMSCs, the antioxidant capacity of AMSCs decreased, but after treatment with Na₂SeO₃, levels of GSH and T-AOC in the AMSCs increased, indicating that Na₂SeO₃ was able to improve the antioxidant capacity of AMSCs. The observed increases in the levels of growth factors, including HGF, SCF, and TGF-β, indicate that the paracrine capacity of AMSCs was also enhanced after treatment with Na₂SeO₃.

The results of both the microarray analysis and quantitative RT-PCR experiments showed that the mRNA expression levels of many genes involved in mitosis, DNA replication and repair, ubiquitination, mitochondrial transport, synthesis and metabolism, cell growth, and migration were altered after supplementation with Na₂SeO₃, including Rlim, Ankrd12, Chid1, Selenbp1, Timm17b, Ankra2, and Tars. However, the specific mechanism by which Na₂SeO₃ involves these genes to impact AMSCs must be further investigated. Additionally, the mechanism by which AMSCs and Na₂SeO₃ affect immune cells in rats with HT is unclear and will be detailed in a separate report.

In conclusion, we found that AMSCs combined with Na₂SeO₃ significantly restored the function and structure of the thyroid, improving immunity in rats with HT. Na₂SeO₃ promoted cell growth and improved the secretory ability of AMSCs. These findings may provide a new approach for HT treatment. However, the specific effects and mechanisms must be further investigated.

The datasets used and/or analyzed during the current study are available from the corresponding author upon request.

Disclosure of conflict of interest

None.