The Effects Of Jiayankangtai Granules(JYKT) On Autoimmune Thyroiditis Rats Based On Transcriptome Sequencing

ObjectiveTranscriptomics was used to analyze the protective effect of Jiayan Kangtai(JYKT)granules on autoimmune thyroiditis(AIT)in rats and its possible mechanismMethodsForty AIT model rats were established following high iodine induction and injection of porcine thyroglobulin adjuvant into Lewis rats.After the successful establishment of the AIT model in rats,according to the concentration of serum TPOAb,the rats were randomly divided into a model group,a JYKT low-dose group(0.708g/kg),a JYKT middle-dose group(1.417g/kg)and a JYKT high-dose group(2.834g/kg),with 10 rats in each group.Rats in the normal group and the model group were given lml/100g double distilled water,and rats in JYKT three-dose group were given JYKT granules for 8 weeks.Samples were taken after drug intervention,and the concentrations of T3,T4,FT3,FT4,TSH.TGAb,TPOAb and TRAb in rat serum were detected.Unilateral thyroid lobes was taken with ultra-low temperature and fixed in 4%paraformaldehyde for HE staining and immunohistochemical detection.The other thyroid lobes was quickly put into liquid nitrogen,and RNA was extracted for transcriptome sequencing and RT-PCR detection.Detection of JYKT components by LC/MS.The expression levels of Jak2,STAT3 and HIF-1α mRNA in thyroid tissue were detected by RT-PCR Immunohistochemistry was used to detect the changes of p-Jak2,p-STAT3 and HIF-1αIOD/Area in thyroid tissueMethods of cell experiment:Twenty female Lewis rats were randomly divided into the normal group(given double distilled water)and JYKT group(high-dose granules 2.834g/kg),with 10 rats in each group.After 7 days of continuous administration,blood was collected by anesthesia to obtain drug-containing serum.An autoimmune thyroiditis cell injury model was established by IFN-y intervention in Nthy-ori 3-1 cells for 24 hours.After intervention with drug-containing serum,CCK-8 method was used to detect cell activity.Immunofluorescence and Western blot were used to detect fluorescence intensity and protein expression of p-Jak2.p-STAT3 and HIF-1α,respectively to verify the results of animal experiments.Results1.Determination of JYKT by LC/MS:As a result,10122 metabolites were obtained,including 1870 negative ions and 8252 positive ions with the top 10 metabolites.2.Thyroid function examination:Compared with the normal group,T3,T4,FT3,FT4 in the model group increased significantly(P<0.01),while TSH decreased(P<0.01).Compared with the model group,after intervention by JYKT,T3,T4,FT3,FT4 decreased and TSH increased in the low-dose group,but there was no significant difference.In the middle-dose group,T3(P<0.05),FT3(P<0.01)and FT4(P<0.01)decreased significantly,but TSH increased without statistical difference.In the high-dose group,T3,T4,FT3,FT4 decreased and TSH increased significantly(P<0.01).3.Detection of thyroid autoantibodies:Compared with normal group,TGAb,TPOAb and TRAb in model group increased significantly(P<0.01).Compared with the model group.TGAb and TPOAb in the low and middle dose groups were significantly decreased(P<0.01).TGAb,TPOAb and TRAb were all decreased in high dose group,with statistical difference(P<0.01).4.Morphological detection:In the normal group,a large number of intact thyroid follicles were seen,the red colloid in the cavity was filled evenly,and the follicular epithelial cells were intact,while in the model group,the thyroid follicular stroma showed diffuse lymphocyte infiltration,and a large number of follicular cavities were destroyed or reduced.The distribution of colloid in the cavity is uneven or reduced,and the follicular wall becomes thinner and destroyed.The infiltration of lymphocytes in the thyroid follicular stroma in the three treatment groups was significantly less than that in the model group,the follicular epithelial cells were more complete,and the colloid content was slightly lower than that in the model group,but slightly less than that in the model group.The high dose group was slightly better than the low and middle dose groups.5.Transcriptome detection:By sequencing,it was found that JYKT could produce 49 differentially expressed genes with autoimmune thyroiditis rats,including 16 down-regulated genes and 33 up-regulated genes.Two immunologically related differentially expressed genes,Dnase113 and JAK3,were obtained from GO enrichment analysis.JYKT may alleviate the inflammatory reaction and related immune reaction in thyroid tissue by up-regulating Dnase113 and down-regulating JAK3 expression in thyroid tissue.In KEGG enrichment analysis,hypoxia inducible factor HIF-1 signal pathway has the highest enrichment score in the JYKT group compared with the model group,and there are obvious differences.In addition,Jak-STAT signaling pathway,epidermal growth factor receptor signaling pathway.primary immunodeficiency and necrotic apoptosis are all related to autoimmune thyroiditis6.PT-PCR detection:Compared with the normal group,the relative expressions of Jak2.STAT3 and HIF-1α mRNA in the model group were significantly increased(P<0.01)Compared with the model group,Jak2(P<0.05),STAT3(P<0.01)and HIF-1α(P<0.05)were all down-regulated in the JYKT high-dose group.7.Immunohistochemical detection:Compared with normal group,there were more p-Jak2,p-STAT3 and HIF-1α protein expressions in thyroid tissue of model group rats(P<0.01)Compared with the model group,the expression of p-Jak2,p-STAT3 and HIF-1α protein decreased in the JYKT high-dose group(P<0.05)8.Nthy-ori 3-1 cell detection results:Thyroid cell activity in the JYKT group was significantly higher than that in the model group.Immunofluorescence and Western blot showed that compared with normal group,the expression values of p-Jak2/Jak2,p-STAT3/STAT3 and HIF-1α/β-actin in model group were significantly up-regulated(P<0.01).and fluorescence intensity also increased(P<0.01).Compared with the model group,the expression values of p-Jak2/Jak2,p-STAT3/STAT3 and HIF-1α/β-actin were down-regulated(P<0.05 or P<0.01)and the corresponding fluorescence intensity were also decreased(P<0.05 or P<0.01)in the JYKT groupConclusions1.JYKT can correct thyroid function and alleviate thyroid structural damage to a certain extent,which may be related to its reduction of serum TGAb,TPOAb and TRAb concentrations.2.JYKT may alleviate the inflammatory reaction and related immune reaction in thyroid tissue by up-regulating Dnase113 and down-regulating JAK3 mRNA expression in thyroid tissue;Meanwhile,JYKT may down-regulate the expression of HIF-1α signal pathway,Jak-STAT signal pathway,primary immunodeficiency signal pathway and necrotic apoptosis signal in thyroid lobes,so as to alleviate the development of AIT.3.JYKT may inhibit Jak2/STAT3/HIF-1α pathway to alleviate the development of AIT4.JYKT has a certain protective effect on thyroid cells,which may be related to the down-regulation of the expression of p-Jak2/Jak2,p-STAT3/STAT3 and HIF-1α/β-actin,and the corresponding fluorescence intensity,suggesting that JYKT has a definite inhibitory effect on the Jak2/STAT3/HIF-1α pathway.