Study On The Regulation Of MiRNAs Expression By Buzhong Yiqi Decoction In Mice With Autoimmune Thyroiditi

ObjectiveThe aim of this study was to investigate the effect of Buzhong Yiqi decoction（BZYQD）on the miRNA expression profile of thyroid tissue in autoimmune thyroiditis（AIT）mice and to elucidate the possible mechanism of BZYQD in intervening the inflammatory infiltration status of thyroid tissue in AIT mice.This study aimed to provide experimental evidence for the clinical application of BZYQD in the treatment of AIT.Materials and MethodsThirty NOD.H-2^h4 mice were randomly divided into three groups:the normal control group（NC group）,the model group（MG group）,and the BZYQD group（BG group）,with ten mice in each group.AIT models were established by administering0.05%sodium iodide to the mice.The BG group was administered BZYQD(9.56g·kg^-1)by gavage,while the NC and MG groups were given an equal amount of distilled water by gavage once daily for eight weeks.After the intervention,thyroid tissue and serum were collected from each group of mice.The pathological changes in the thyroid tissue of the mice were observed by HE staining,and the levels of thyroglobulin antibody（TgAb）,interleukin（IL）-1β,IL-6,and IL-17 in the serum were detected by enzyme-linked immunosorbent assay（ELISA）.The differentially expressed miRNAs in the thyroid tissue of the mice were verified by RNA sequencing（RNA-seq）technology and subjected to bioinformatics analysis.The expression levels of miR-326-3p,miR-128-3p,miR-223-5p,miR-141-3p,miR-871-3p,and miR-204-3p in the thyroid tissue of each group of mice were detected by real-time quantitative PCR（RT-q PCR）assay.Results1.Histopathological changes of mouse thyroid tissue and expression levels of TgAb:In the NC group,the thyroid follicular epithelium appeared cuboidal,the follicles were abundant and closely arranged.In the MG group,the thyroid follicular epithelial cells were flattened,the colloid in the follicular lumen was uneven,and lymphocyte infiltration was visible in the stroma.In the BG group,the thyroid follicular epithelial cells were relatively intact,the colloid content was slightly reduced,and the degree of lymphocyte infiltration was reduced.Compared with the NC group,the MG group showed obvious infiltration of inflammatory cells in the thyroid tissue,and the serum TgAb level was significantly increased（P<0.01）.Compared with the MG group,the BG group showed a reduction in the degree of inflammation in the thyroid tissue and a decrease in serum TgAb level（P<0.01）.2.Expression levels of inflammatory factors in mouse serum:Compared with the NC group,the expression levels of IL-6 and IL-17 in the MG group were significantly increased（P<0.01）,and the expression level of IL-1βwas significantly decreased（P<0.05）.Compared with the MG group,the expression levels of interleukin-1β（IL-1β）,interleukin-6（IL-6）,and interleukin-17（IL-17）in the BG group were significantly decreased（P<0.05）.3.Influence of miRNA expression profile in mouse thyroid tissue:Compared with the NC group,the MG group showed 154 differentially expressed miRNAs,among which miR-326-3p,miR-128-3p,miR-223-5p,miR-8120 were significantly upregulated,while miR-141-3p,miR-871-3p,miR-204-3p,miR-679-5p were significantly downregulated.Compared with the MG group,the BG group showed112 differentially expressed miRNAs,among which miR-128-3p,miR-690,miR-326-3p,miR-6715-5p were significantly downregulated,while miR-123-3p,miR-871-3p,miR-141-3p,miR-204-3p,miR-374c-3p were significantly upregulated.RT-q PCR results showed that the expression of miR-326-3p,miR-128-3p,miR-223-5p,miR-141-3p,miR-871-3p,miR-204-3p was consistent with the sequencing results.The Buzhong Yiqi decoction can regulate various miRNAs related to target genes,such as immune cell cytokines and inflammation-related molecules.Bioinformatics analysis of the target genes of miRNAs showed GO functional enrichment in T cell activation regulation,oxidative stress,miRNA binding,etc.;KEGG pathway enrichment in phosphatidylinositol-3 kinase（P13K）/protein kinase B（AKT）signaling pathway,mitogen-activated protein kinase（MAPK）signaling pathway,cyclic adenosine monophosphate（c AMP）signaling pathway,etc.Three key genes were predicted by differentially expressed miRNAs:Mothers Against DPP Homolog 3（Smad3）,Janus Kinase 2（Jak2）,Signal Transducer and Activator of Transcription 3（Stat3）.Conclusion1.Buzhong Yiqi decoction can alleviate the inflammation infiltration of thyroid lymphocytes,reduce serum TgAb and immune cytokines（IL-1β,IL-6,IL-17）in AIT mice,and relieve the immune imbalance state of AIT.2.Buzhong Yiqi decoction can regulate the miRNA expression profile of thyroid tissue in AIT mice,intervene in the expression of various miRNAs such as miR-326-3p,miR-128-3p,miR-223-5p,miR-141-3p,miR-871-3p,and miR-204-3p.The differentially expressed miRNAs regulate some target genes involved in various inflammatory signaling pathways,which may be one of the mechanisms by which Buzhong Yiqi decoction intervenes in the immune imbalance state of AIT mice.