Study On The Role Of GLIS3 In Thyroid Injury Induced By Real-ambient PM_2.5 Exposure In Mice

Objective:Epidemiological studies shown that PM_2.5 can interfere with thyroid function,but the effect of PM_2.5 on thyroid function is controversial,and there is a lack of experimental studies on the mechanism of thyroid injury caused by PM_2.5.In this study,a real-ambient PM_2.5 exposure system was constructed to clarify the thyroid damage effect of PM_2.5exposure in mice,and to explore the mechanism of GLI-like zinc finger protein（GLIS3）in thyroid injury induced by PM_2.5 exposure,so as to provide scientific basis for the prevention and treatment of thyroid injury caused by PM_2.5 exposure.Methods:1.C57BL/6J mice were exposed to Shijiazhuang real environment exposure system for4 or 8 weeks for 16 hours a day.During the exposure period,the mice ate freely and maintained the rule of 12 hours day/12 hours night.The concentration of PM_2.5 in cage was measured at the same time in the morning,middle and evening every day.2.At the end of exposure,the thyroid tissues of mice were taken for Hype staining.The level of thyroid hormone in serum was determined by ELISA test;Determination of urinary iodine level in mice by urinary iodine kit;The expression level of inflammatory factors in thyroid tissue was detected by RT-PCR,and the expression of thyroid hormone synthesis related genes and GLIS3 in thyroid tissue were determined by RT-PCR,Western blotting and immunohistochemistry.Differential genes were screened by transcriptome sequencing to explore the possible mechanism of thyroid injury induced by PM_2.5.Results:1.Thyroid tissue pathological damage and functional disturbance were caused by PM_2.5exposure.After 4 and 8 weeks of PM_2.5 exposure,the thyroid follicular cells became smaller,the follicular epithelial cells increased,the colloid content in the follicular cavity decreased,and the free inflammatory cells increased.After 8 weeks of PM_2.5 exposure,the level of free tetraiodothyronine（FT4）increased significantly,while the levels of thyroid stimulating hormone（TSH）,thyroid peroxidase antibody（TPO-Ab）and thyrotropin receptor antibody（TR-Ab）decreased significantly（P<0.05）.2.PM_2.5 increases the expression of thyroid synthesis-related genes and transcription factors.The m RNA expressions of sodium iodide transporter（NIS）,thyroid peroxidase（TPO）and thyroglobulin（TG）genes related to thyroid hormone synthesis were significantly increased after 8 weeks of PM_2.5 exposure（P<0.01）.The protein expression levels of NIS,TPO and TG increased significantly after 4 and 8 weeks of PM_2.5 exposure（P<0.01）.After 8 weeks of PM_2.5 exposure,GLIS3 and paired box 8（PAX8）m RNA were significantly up-regulated,and the protein expressions of GLIS3 and PAX8 were significantly increased after 4 and 8 weeks of PM_2.5 exposure（P<0.01）.3.PM_2.5 exposure leads to the activation of Rap1/PI3K/AKT signal pathway.In transcriptional group,KEGG analysis of differential genes showed that the genes of Ras-like small GTPases（Rap1）signal pathway were significantly down-regulated,and Rap1+GTP was closely related to the activation of transcription factors.The results showed that the protein expression levels of Rap1,PI3K and AKT were significantly increased（P<0.05）.Conclusion:1.PM_2.5exposure caused thyroid tissue damage,caused thyroid pathological structure changes,induced abnormal thyroid hormone levels and interfered with thyroid function,suggesting that PM_2.5 exposure could lead to changes in thyroid structure and function.2.PM_2.5 exposure significantly upregulated the expression of thyroid hormone synthesis-related genes NIS,TPO and TG in mice,thus promoting the synthesis of TH.Further in-depth analysis of thyroid transcription factor expression level,found that PM_2.5 can significantly increase the expression of GLIS3,suggesting that PM_2.5 can activate GLIS3to promote thyroid hormone synthesis gene up-regulation,resulting in abnormal TH synthesis.3.Transcriptome sequencing and further verification analysis showed that the expression of Rap1,PI3K and AKT genes increased significantly after PM_2.5 exposure,suggesting that PM_2.5 can activate Rap1/PI3K/AKT pathway,up-regulate GLIS3 expression,induce thyroid hormone synthesis gene expression,and disrupt the regulation of TH homeostasis.