二氢睾酮对Graves病小鼠模型共刺激分子表达的影响

doi:10.24920/003594

Abstract

Objective Graves’ disease is the most common autoimmune thyroid disease and its prevalence and clinical manifestations are disparate between females and males. Costimulatory molecules play an essential role in regulating autoimmune responses. The objective of this study was to determine if expression of inhibitory molecules was correlated with treatment by dihydrotestosterone (DHT) in an in vivo BALB/c mouse model of experimental autoimmune Graves’ disease.
Methods Female BALB/c mice were immunized three times with thyroid stimulating hormone receptor A-subunit encoded by adenovirus to establish a Graves’ disease model. Three different doses of DHT or a matching placebo were administered by implantation of slow-release pellets a week before the first immunization. Four weeks after the third immunization, the mice were euthanatized, and then the spleen and thymus were removed. Total thyroxine and free thyroxine levels in serum of mice were detected using a radioimmunoassay kit. Real-time polymerase chain reaction was performed to estimate the expression of costimulatory molecules in lymphocytes from the spleen and thymus. Flow cytometry was used to analyze the percentage of CD4⁺ T cells in splenic lymphocytes. Quantitative data were compared with unpaired t-tests. Correlation between two variables was analyzed using Analysis of Variance.
Results Treatment with DHT can dramatically reduce total thyroxine and free thyroxine levels. Higher expression of programmed death-1 was found in the spleen of Graves’ disease mice receiving 5 mg of DHT treatment (0.635±0.296 vs. 0.327±0.212; t=2.714, P=0.014), similarly, T-cell immunoglobulin domain and mucin domain 3 (TIM-3) in both the spleen (1.004±0.338 vs. 0.646±0.314; t=2.205, P=0.022) and the thymus (0.263±0.127 vs. 0.120±0.076; t=3.221, P=0.004) also increased after 5 mg of DHT treatment compared with the parallel placebo model mice. Moreover, the percentage of CD4⁺ T cells declined in the splenic lymphocytes of Graves’ disease mice treated with 5 mg of DHT (19.90%±3.985% vs. 24.05%±2.587%; t=2.804, P=0.012). A significant negative association was observed between expression of TIM-3 in the spleen and serum levels of total thyroxine (r=-0.7106, P=0.014) as well as free thyroxine (r=-0.6542, P=0.029).
Conclusion This study demonstrates that DHT can ameliorate experimental autoimmune Graves’ disease, which may occur by up-regulating expression of programmed death-1 and TIM-3 and inhibiting development of CD4⁺ T cells.

Key words: Graves’ disease, 5α-dihydrotestosterone, costimulatory molecules, inhibitory molecules

Liu Lianye, Shi Bingyin, Zhao Fengyi, Hou Peng, Liu Shu, Liu Xiaomei, Wu Liping. Effect of Dihydrotestosterone on CostimulatoryMolecules in a Mouse Model of Graves’ Disease[J].Chinese Medical Sciences Journal, 2020, 35(3): 215-225.

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Genes	Forward primers	Reverse primers	Fragment size (bp)
CTLA-4	5’-TGGGTTCAAACACATCTCAAGGC-3’	5’-TGTCGTGGCACAGACCTCAG-3’	349
BTLA	5’-GCCATTCAGTAACCATCCATGTG-3’	5’-GCAAGGTGTAAAGCAGCCAAGTC-3’	150
PD-1	5’-CCGCTTCCAGATCATACAG-3’	5’-CTCTGGCCTCTGACATACTTG-3’	329
TIM-3	5’-AGCCCATGTGGAAATTTTTG-3’	5’-CTCCAAGAACCCTAACCACG-3’	95
CD28	5’-GTGGTTGCTGGAGTCCGTT-3’	5’-GCTGGTAAGGCTTTCGAGTG-3’	151
ICOS	5’-GTCAAACAACAGCGTCTC-3’	5’-TGCAAATATCCTCCACTAA-3’	129
OX40	5’-GTAGACCAGGCACCCAACC-3’	5’-GGCCAGACTGTGGTGGATTGG-3’	268
CD154	5’-ACGTTGTAAGCGAAGCCAAC-3’	5’-TATCCTTTCTTGGCCCACTG-3’	61
GAPDH	5’-CATGACCACAGTCCATGCCAT-3’	5’-TCAGATCCACGACGGACACATT-3’	215

Groups	n	TBI^a (%)	TT4 (nmol/L)	FT4 (pmol/L)
Control group	12	0.67±6.91	38.22±10.18	4.13±2.02
EAGD group	10	80.30±11.31	159.33±76.58	18.43±11.21
t value		20.31	5.446	4.354
P value		<0.0001	<0.0001	0.0003
Placebo_{1.5 mg DHT}	10	79.10±11.13	130.37±62.03	15.57±12.26
1.5 mg DHT group	11	70.36±10.01	75.29±35.78	11.09±5.02
t value		1.894	2.524	1.116
P value		0.07	0.02	0.28
Placebo_{5 mg DHT}	10	81.50±7.75	154.31±71.69	16.69±12.24
5 mg DHT group	11	70.00±20.09	78.12±32.18	6.831±3.02
t value		1.696	4.962	2.554
P value		0.11	0.01	0.02
Placebo_{15 mg DHT}	9	80.11±11.24	136.81±72.20	17.89±12.16
15 mg DHT group	11	69.82±12.81	75.80±53.15	6.90±4.38
t value		1.972	2.176	2.799
P value		0.06	0.04	0.01

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