| Objective:Hashimoto’s thyroiditis is a classic representative of autoimmune thyroiditis,and the incidence is gradually increasing.Studies have confirmed that the Notch signaling pathway can participate in the occurrence and development of a variety of autoimmune diseases by regulating the levels of Treg/Th17 cytokines.Therefore,Notch protein can also promote the occurrence and development of Hashimoto’s thyroiditis by affecting the levels of Treg/Th17 cytokines.We carried out clinical trials to observe the clinical efficacy of Xiaoyingdaotan Decoction,created a mouse model of Hashimoto’s thyroiditis,induced thyroid inflammatory cell lines,and detected Notch protein expression and Treg/Th17 cytokines,the mechanism of the Chinese medicine Xiaoyingdaotan Decoction on Hashimoto’s thyroiditis was explored in depth to provide a scientific basis for its clinical application.Methods:A total of 66 patients with Hashimoto’s thyroiditis with stagnation of qi and phlegm and coagulation syndrome were collected to observe the clinical efficacy of Chinese medicine Xiaoyingdaotan Decoction.The patients were scored for TCM symptoms before,during and after treatment.Serum thyroid function,Notch protein expression,and Treg/Th17 cytokine levels were detected by ELISA before and after treatment.Thyroid ultrasound was performed before and after treatment.A mouse model of Hashimoto’s thyroiditis was created.After the treatment,the serum thyroid function,Notch protein expression and Treg/Th17 cytokine levels were detected by ELISA.At the same time,the thyroid inflammatory cell line was induced,and the serum containing the Chinese medicine Xiaoyingdaotan Decoction was used to act on the cells,after the experiment,Real-time qPCR and Western blot were used to detect the expression of Notch protein in each group of cell genes,and ELISA method was used to detect the levels of Treg/Th17 cytokines and inflammatory factors in each group.Results:1.Results of clinical trials:From the perspective of the efficacy of TCM syndromes and diseases,the treatment group was better than the control group,with significantly statistical differences(P<0.001);the serum Notch protein expression level of HT patients was significantly higher than that of healthy people,and the difference was significant(P<0.001);the expression level of serum Notch protein in the treatment group was significantly lower than that in the control group after treatment,with significant differences(P<0.001).After treatment,serum Treg cell key activation protein levels in the treatment group were up-regulated compared to before treatment,with significant differences(P<0.05).Compared between the groups,in the treatment group after treatment,the serum FOX-P3 level was significantly higher than that in the control group(P<0.05),but there was no significant difference in the levels of TGF-β and IL-10 between the two groups(P>0.05).After treatment,the levels of STAT3,IL-17,and IL-22,the key activating proteins of Th17 cells in the treatment group,were lower than before treatment,with significant differences(P<0.05).In comparison between groups,in the treatment group,the levels of STAT3 and IL-17 after treatment were lower than control group(P<0.05),but there was no significant difference in the levels of RORyt and IL-22 between the two groups(P>0.05).In the middle of treatment,in the treatment group,the decrease of TSH was more obvious than that of the control group,with statistical difference(P<0.05),but after treatment,there was no statistical difference in TSH levels between the two groups(P>0.05).After treatment,compared with the control group,the serum TPOAb level of the treatment group decreased significantly(P<0.05),but there was no statistical difference in the FT3、FT4、TGAb level of the two groups of patients(P>0.05).2.Animal experiment results:Compared with the model group,the serum TSH of mice in the western medicine control group,Chinese medicine low-dose group,Chinese medicine high-dose group and Notch protein inhibitor group decreased significantly,with statistical difference(P<0.05).Compared with the model group,the levels of FT3、FT4 did not change,and serum thyroid autoantibody TGAb and TPOAb of the other groups of mice did not significantly decrease,with no statistical difference(P>0.05).Compared with the model group,the serum Notch protein expression of mice was significantly higher than other groups,with significantly statistical differences(P<0.001).Comparing the high-dose Chinese medicine group with the western medicine control group,the low-dose Chinese medicine group,and the model group,the level of the key activation protein FOX-P3 in serum of Treg cells in mice was significantly increased,with statistical differences(P<0.05).The serum TGF-β levels of the three groups of traditional Chinese medicine and the Notch protein inhibition group were significantly higher than model group and western medicine group,with statistical differences(P<0.05).The serum IL-10 levels of mice in each group were not statistically different(P>0.05).The level of the key activation protein of Th17 cells compared with the model group,the serum STAT3,RORγt,and IL-22 levels of the other groups of mice were significantly reduced(P<0.05).There was no statistical difference in serum IL-17 levels of mice in each group(P>0.05).3.Cell experiment results:The expression level of Notch protein in the 5 groups of thyroid inflammatory cell genes was compared.Compared with the other three groups,the iNotch+DS group and DS group showed that the β-actin value of Notch protein in the thyroid inflammatory cell genes was significantly down-regulated,with statistical differences(P<0.05).The levels of key Treg cell activation proteins FOX-P3,TGF-β and IL-10 in the cell supernatants were compared in pairs.Compared with the other three groups,the key activation proteins of Treg cells were significantly up-regulated in the iNotch+DS group and DS group(P<0.001).The levels of key Th17 cell activation proteins STAT3,IL-17 and IL-22 in the cell supernatants were compared,the iNotch group,iNotch+DS group,and DS group were significantly down-regulated compared with the Blank group and NC group,with significantly statistical differences(P<0.001),but no statistical difference in the level of RORγt among the groups(P>0.05).The levels of inflammatory factors in the cell supernatant were compared in pairs.The inflammatory factors TNF-α,IL-1β,and IL-6 in the cell supernatant of the iNotch group,iNotch+DS group,and DS group were significantly lower than those in the Blank group and NC group,the difference was significant(P<0.05).The level of the inflammatory factor COX-2 in the cell supernatant of the iNotch+DS group was significantly lower than that of the NC group,and the difference was significant(P<0.05).There was no significantly statistical difference in the levels of inflammatory factor IL-8 in the cell supernatant(P>0.05).Conclusion:Chinese medicine Xiaoyingdaotan Decoction has significant therapeutic effects on HT patients with stagnation of Qi and phlegm and HT mouse models.The mechanism may be related to the regulation of Notch protein pathway mediated immune inflammatory response of Treg/Th 17 cell axis. |
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