Mechanism research of the modified Xiaoyao Powder on inflammatory response of rats with syndrome of stagnation of liver qi and spleen deficiency of experimental autoimmune thyroiditis based on mirRNA326 regulating Th17 cells

Yinan LI; Jingxue SHEN; Lan ZHANG

ABSTRACT

Objective:

To explore the mechanism of modified Xiaoyao Powder on inflammatory response of rats with syndrome of stagnation of liver qi and spleen deficiency of experimental autoimmune thyroiditis (EAT) from the perspective of differentiation of microrna 326 (miR326) regulating Th17 cell.

Methods:

48 rats were randomly divided into normal group (12 rats) and model group (36 rats) respectively and they were immunized twice a week with high iodine water combined with subcutaneous injection of thyroglobulin. From the fifth to eighth weeks, 36 rats were immunized once a week. From the fifth week, the model group with liver depression and spleen deficiency syndrome of Traditional Chinese Medicine was reproduced with chronic restraint stress, excessive fatigue and eating incoherence methods. The modelrats were randomly divided into model group, Xiaoyao Powder group and Jinshuibao group. Rats in Xiaoyao Powder group were gavaged with 13.63 g/(kg·d) Xiaoyao Powder modified granule suspension, and rats in Jinshuibao group were gavaged with 477 mg/(kg·d) Jinshuibao suspension, twice a day, for 8 weeks.The levels of serum FT3, FT4, TSH, TGAb and TPOAb were detected by ELISA; the expression of miR326, IL-17 mRNA, IL-4 mRNA and IFN-γ mRNA were detected by PCR. The expression of Ets-1 protein in thyroid tissue was detected by Wes method, and the proportion of CD4 + IFNγ + T cells, CD4 + IL-4 + T cells and CD4 + IL-17 + T cells were detected by flow cytometry, HE staining was used to detect the pathological manifestations of thyroid tissue in each group.

Results:

Compared with the model group, the serum TSH [(3 328.88±724.45) pg/ml vs. (1 900.25±203.91) pg/ml] in Xiaoyao Powder group increased ( P<0.01), TGAb [(63.60±9.01) IU/ml vs. (96.19±10.74) IU/ml] and TPOAb [(6.84±1.45) IU/ml vs. (11.62±2.06) IU/ml] decreased ( P<0.01), and the expression of miR326 (3.57±0.57 vs. 7.63±0.90),IL-17 mRNA (6.71±0.97 vs. 13.02±1.18) significantly decreased ( P<0.01), the expression of Ets-1 (0.71±0.40 vs. 0.39±0.02) significantly increased ( P<0.01), the ratio of CD4 +IFN-γ + T cell [(13.10±2.23)% vs. (20.7±2.07)%], CD4 +IL-17 + T cell ratio [(18.90±1.31)% vs. (25.1±1.03)%] significantly decreased ( P<0.01), and thyroid histopathology changed significantly.

Conclusion:

Modified Xiaoyao Powder could regulate the expression of target protein Ets-1 upward, inhibit the differentiation of Th17 cells and further reduce the expression of IL-17 mRNA by regulating the expression of mir-326 downward in the thyroid tissue of EAT rats, so as to improve the inflammatory response of rats with liver depression and spleen deficiency.