| Objective:This study aims to observe the effect of herb-partitioned moxibustion(HPM)on mi RNA expression profile of thyroid tissue in EAT rat,and to explore the immune regulation mechanism of HPM on EAT rats.We want to clarify that HPM may regulate the expression of mi RNAs of EAT rats,then inhibit the expression of immune cytokines and the activation of inflammatory signaling pathways,finally reduce the release of inflammatory mediators,and plays a role in improving thyroid autoimmune inflammation,in order to provide more theoretical basis for the clinical application of HPM treatment on HT.Methods:Female SD rats of clean grade weighing 100±20g were randomly divided into normal control(NC)group and modeling group.EAT model rats were established by combined immunization of porcine thyroglobulin emulsified with complete and incomplete freund’s adjuvant and iodine.After the model was completed and successfully identified,the model rats were randomly divided into three groups: EAT model(EAT)group,herb-partitioned moxibustion(HPM)group and western medicine(Med)group.Rats in the HPM group were treated with corresponding treatment at Dazhui(DU14),Mingmen(DU4)and Tiantu(RN22),Guanyuan(RN4)acupoints alternately,once daily for consecutive 30 days;Rats in the Med group were treated with levothyrocine(1μg/2ml)by gavage,once a day for consecutive 30 days.After intervention,serum and thyroid tissues of each group rats were taken for further detection.HE staining was used to observe the histopathological changes of thyroid tissues;serum TGAb,TPOAb,FT3,FT4,TSH,IL-6 and TNF-α concentration were detected by ELISA;mi RNA high-throughput sequencing was used to detect changes of mi RNA expression profiles in thyroid tissues,then predicted target genes of differentially expressed mi RNA,performed bioinformatics analysis of functional annotation and KEGG pathway,and the differentially expressed mi RNAs that regulated by HPM were verified;real-time quantitative PCR(RT-q PCR)was used to detect the m RNA expression of IFN-γ,IL-4,IL-6,Foxp3,GATA3,IL-17,IL-23,IL-23 R in the thyroid tissues;immunohistochemistry was used to detect the expression of TLR3,TLR4,HMGB1,RAGE,HSP60,My D88,TRIF,IRAK1,TRAF6 and NF-κBp65 proteins.Results:1.Histopathology observation of thyroid tissue: Under light microscope,the thyroid follicular structure in the NC group was clear and in a circle or oval shape,the follicular cavity was filled with colloid,and no lymphocyte infiltration was observed.In the EAT group,the thyroid follicular structure was extensively damaged and atrophied,and numerous lymphocytes were observed,with interstitial hyperplasia of numerous fibrous tissues.The thyroid tissue morphology in the HPM group was improved compared with the EAT group,the shape of thyroid follicles was slightly regular,and a small amount of thyroid follicles were destroyed,with a small amount of lymphocytes infiltrated and fibrous hyperplasia in the interstitial.The thyroid follicular structure in the Med group was damaged,and a small amount of lymphocyte infiltration and fibrous hyperplasia were observed.2.Effect of herb-partitioned moxibustion on EAT thyroid antibody and thyroid function: Thyroid antibody: Compared with the NC group,the concentration of serum TPOAb and TGAb in the EAT model group were increased(both P<0.01);compared with the EAT model group,the concentrations of serum TPOAb and TGAb in the HPM group were significantly decreased(both P<0.05).Thyroid function: Compared with the NC group,the serum TSH concentration in the EAT model group was increased,while the concentrations of FT3 and FT4 were decreased,the difference was statistically significant(all P<0.01);compared with the model group,the concentration of serum TSH in the HPM and Med group was significantly decreased(respectively P<0.05,P<0.01);the concentrations of serum FT3 and FT4 in the Med group were significantly increased(respectively P<0.01,P<0.05).3.Effect of herb-partitioned moxibustion on mi RNA expression profile of EAT thyroid: Compared with NC group,the expression of various mi RNAs in thyroid tissue of EAT model rats were changed,including that the expression of mi R-133a-3p,mi R-3084b-3p,mi R-3084 d,mi R-133b-3p,mi R-3084a-3p,mi R-1249,mi R-365-3p,mi R-133a-5p,mi R-1b,mi R-129-2-3p,mi R-346,mi R-1-3p,mi R-664-3p,mi R-326-3p,mi R-149-5p,mi R-206-3p,mi R-328b-3p were significantly up-regulated,while the expression of mi R-547-3p,mi R-298-5p,mi R-296-3p,mi R-331-5p were down-regulated.After herb-partitioned moxibustion treatment,the expression of mi R-346 was significantly down-regulated,and the expression of mi R-331-5p was significantly up-regulated.In the Med group,the expression of mi R-133b-3p,mi R-133a-3p,mi R-1b,mi R-1-3p,mi R-3084 d,mi R-3084a-3p and mi R-3084b-3p were significantly down-regulated,and the expression of mi R-331-5p was significantly up-regulated.The target genes of mi RNAs regulated by HPM include a variety of genes related to the pathogenesis of HT,such as transcription factor,immune cytokine,and inflammatory signaling pathway-related moleculars,and with many biological functions of protein binding,cellular process,cellular,material metabolic process,biological regulation and so on.Furthermore,in addition to the more enriched metabolic pathways,MAPK signaling pathway,PI3K-Akt signaling pathway,Ras signaling pathway,endocrine resistance,the KEGG pathway they involved in also included chemokine signaling pathway,thyroid hormone signaling pathway,Toll-like receptor signaling pathway,NF-kappa B signaling pathway and so on.4.Verification of differentially expressed mi RNAs regulated by herb-partitioned moxibustion: Compared with the NC group,the expression of mi RNA-346 in the thyroid tissue of EAT group rat was significantly increased(P<0.01),and the expression of mi RNA-331-5p was significantly decreased(P<0.05).Compared with the EAT group,the expression of mi RNA-346 in the thyroid tissue of HPM group rat was decreased(P<0.05),and the expression of mi RNA-331-5p was increased(P<0.05),the expression of mi RNA-346 in the thyroid tissue of Med group rat was also significantly decreased(P<0.01).5.Effect of herb-partitioned moxibustion on the concentration of serum inflammatory factors and m RNA expression of immune cytokines in thyroid of EAT rats: Compared with the NC group,the serum IL-6 and TNF-α concentration and the expression of IFN-γ,IL-4,IL-6,TGF-β1,IL-17,IL-23,IL-23 R m RNA in the thyroid tissue of EAT model group rats were significantly increased(P<0.01 or P<0.05),while the expression of Foxp3 m RNA was significantly decreased(P<0.01).Compared with the EAT model group,the serum IL-6 and TNF-α concentration and the expression of IFN-γ,IL-4,IL-6,TGF-β1,IL-17,IL-23,IL-23 R m RNA in the thyroid tissue of HPM group rats were decreased,and the expression of Foxp3 m RNA was increased(P<0.01 or P<0.05);the serum IL-6 concentration and the expressions of IL-4,IL-6,TGF-β1,IL-17,IL-23 and IL-23 R m RNA in the thyroid tissue of Med group rats were also significantly decreased(P<0.01 or P< 0.05).6.Effect herb-partitioned moxibustion on the expression of TLR signal-related protein in thyroid of EAT rats: Compared with the NC group,the expression levels of TLR3,HMGB1,RAGE,HSP60,My D88,TRIF,IRAK1,TRAF6 and NF-κBp65 proteins in the thyroid tissue of EAT group were all significantly increased(all P<0.01).Compared with the EAT model group,the expression levels of TLR3,HMGB1,HSP60,My D88,TRIF,IRAK1,TRAF6 and NF-κBp65 proteins in the thyroid tissue of HPM rats were decreased(P<0.01 or P<0.05),and the expression levels of TLR3,HMGB1,My D88,IRAK1 and NF-κBp65 proteins in the thyroid tissue of Med group were also significantly decreased(P<0.01 or P<0.05).Conclusions:1.Herb-partitioed moxibustion can improve the morphology of thyroid tissue in EAT rats,reduce the concentration of serum thyroid autoantibodies and TSH in EAT rats,and alleviate inflammation of thyroid tissue.2.Herb-partitioed moxibustion can influence the mi RNA expression profile in thyroid of EAT rats,and especially down-regulate the expression of mi RNA-346,up-regulate the expression of mi RNA-346,some target genes of differentially expressed mi RNAs and their involved pathways such as Toll-like receptor signaling pathway are closely related to immunity,which may be also one of the mechanisms of herb-partitioed moxibustion treatment on EAT.3.Herb-partitioed moxibustion can significantly reduce the expression of IFN-γ,IL-4,IL-6,TGF-β1,IL-17,IL-23 and IL-23 R m RNA in thyroid of EAT rats,increase the expression of transcription factor Foxp3 m RNA,regulate the imbalance of immune cytokines,as well as inhibit the abnormal activation of TLR signaling pathway,thereby reduce the release of serum IL-6 and TNF-α,which may be an important mechanism of herb-partitioed moxibustion treatment in relieving and/or inhibiting thyroid autoimmune inflammation. |
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