Effects Of Qijian Xiaoying Prescription On The Intestinal Flora And Intestinal Mucosal Barrier In Rats With Autoimmune Thyroiditis Based On TLR4/MyD88/NF-κB Signaling Pathway

Objective: 1.The effects of Qijian Xiaoying Prescription on thyroid inflammation injury and TLR4/My D88/NF-κB signaling pathway in AIT rats were studied by establishing an animal model of autoimmune thyroiditis(AIT).2.In this study,the intestinal tract was used as the entry point to explore the effects of Qijian Xiaoying Prescription on intestinal flora and intestinal mucosal barrier of AIT rats.Methods: The rat models with AIT were established by high-iodine water feeding combined with subcutaneous injection of porcine thyroglobulin and Freund’s adjuvant,and were randomly divided into control group,model group,selenium group,and Qijian Xiaoying prescription low-dose,medium-dose,high-dose group.Starting from the 7th week of experiment,the drugs were given by intragastric administration for six weeks.Specimens were collected after the end of the 12 th week of experiment.The pathological changes of thyroid tissue and colonic mucosa tissue were observed by H&E staining;The ultrastructure of colonic epithelial cells were observed by transmission electron microscope;The FT3,FT4,TGAb,TPOAb,TNF-α,IFN-γ,IL-10 change in serum of rats and s Ig A content in colon tissue were detected by enzyme-linked immunosorbent assay;The m RNA expression of TLR4,My D88,NF-κB p65 in thyroid tissue of each group were detected by RT-PCR technology;The protein expression of TLR4,My D88,IKKβ,p IKKβ,NF-κB p65 in thyroid tissue and ZO-1,Occludin in colon tissue were detected by Western blotting;The changes of intestinal flora were detected by 16 S r RNA high-throughput gene sequencing technology.Results: 1.Effects of Qijian Xiaoying Prescription on thyroid inflammatory injury in rats with autoimmune thyroiditis 1.1 The results of ELISA:The serum FT3,FT4,TGAb,TPOAb,TNF-α,IFN-γ levels in the model group increased.The serum FT3,FT4,TGAb,TPOAb,TNF-α,IFN-γ levels in selenium yeast group and Qijian Xiaoying Prescription low-dose,medium-dose,high-dose group decreased.Each drug could inhibit thyroid function and down-regulate the levels of thyroid autoantibodies and proinflammatory cytokines in AIT rats.1.2 The results of HE staining:In the model group,the thyroid follicle structure was destroyed,the follicular cavity was atrophy,lymphocyte infiltration was seen,and the thyroid tissue inflammation score increased.After the intervention of selenium yeast and different doses of Qijian Xiaoying Prescription,the structural integrity of thyroid follicles was restored to varying degrees,the degree of lymphocyte infiltration was reduced,and the thyroid tissue inflammation score also gradually decreased.The results show that Qijian Xiaoying Prescription could improve the inflammatory injury of thyroid tissue in AIT rats.2.Effects of Qijian Xiaoying Prescription on TLR4/My D88/NF-κB signaling pathway in rats with autoimmune thyroiditis 2.1 The results of RT-PCR: The m RNA expressions of TLR4,My D88,and NF-κB p65 in thyroid tissue of rats in the model group increased,and m RNA expressions of TLR4,My D88,NF-κB p65 in thyroid tissue of the selenium yeast group and Qijian xiaoying prescription medium-dose,high-dose groups decreased.Although the m RNA expression of Qijian xiaoying prescription low-dose group decreased,there was no statistical difference.2.2 The results of western-blotting:The protein expression of TLR4,My D88,IKKβ,p IKKβ,NF-κB p65 in thyroid tissue of the model group increased,and protein expressions of TLR4,My D88,IKKβ,p IKKβ,NF-κB p65 in thyroid tissue of the selenium yeast group and Qijian xiaoying prescription medium-dose,high-dose groups decreased.While the protein expressions of Qijian xiaoying prescription low-dose group decreased,there was no statistical difference.3.Effects of Qijian Xiaoying Prescription on intestinal flora in rats with autoimmune thyroiditis 3.1The results of species diversity:The Shannon,Simpson,chao,and ace indexes of the model group increased,but the differences were not significant,indicating that the species diversity and abundance of the intestinal flora of AIT rats increased,and there was an overgrowth of intestinal flora.The Shannon,Simpson,chao,and ace indexes of the selenium yeast group increased,while the Shannon,Simpson,Chao,and Ace indexes of the Qijian xiaoying prescription low-dose,medium-dose,high-dose group decreased.The control group and the model group were significantly separated at the PC1 level.The distance between the selenium yeast group and Qijian xiaoying prescription low-dose,medium-dose,high-dose group from the model group gradually increased at the PC1 level.It shows that the composition and structure of the intestinal flora of rats in each group have changed significantly.3.2 Phylum level species composition:At the Phylum level,the intestinal flora of each group mainly includes Firmicutes,Bacteroidetes,Proteobacteria,Tenericutes and Actinomycetes Actinobacteria and others,the Firmicutes,Bacteroidetes and Proteobacteria are dominant bacteria phyla.In the model group,the ratio of Firmicutes and Firmicutes to Bacteroidetes(F/B)increased,and the ratio of Bacteroidetes decreased.The ratio of Firmicutes and F/B value of Firmicutes in the selenium yeast group and Qijian xiaoying prescription lowdose,medium-dose,high-dose group decreased,while the Bacteroidetes increased.3.3 Genus level species composition:At the genus level,the intestinal flora of each group of rats mainly includes Muribaculaceae,Ruminococcaceae,Lachnospiraceae,Lactobacillus,Ba cteroides,Christensenellaceae,Prevotellaceae,Romboutsia,etc,whi ch Lactobacillus and Prevotellaceae changed significantly.In the model group,the abundance of Lactobacillus and Prevotellaceae decreased.The abundance of Lactobacillus in the selenium yeast group and Qijian xiaoying prescription low-dose,medium-dose group increased to varying degrees.However,the abundance of Lactobacillus in Qijian xiaoying prescription high-dose group showed a downward trend,but there was no statistical difference.The abundance of Prevotellaceae in the selenium yeast group and Qijian xiaoying prescription medium-dose,high-dose group showed a downward trend,while the composition of Qijian xiaoying prescription low-dose increased,and there was no statistical difference.3.4 The results of Metabolic pathway prediction: The metabolic functions of intestinal microbes in each group are roughly similar Except for unknown functions,the remaining metabolic functions mainly include carbohydrate transport and metabolism,amino acid transport and metabolism,translation of ribosomal structure and biogenesis,DNA replication and recombination and repair,energy production and Transformation,inorganic ion transport and metabolism,nucleotide transport and metabolism,signal transduction mechanism,coenzyme transport and metabolism and other functions.4.Effects of Qijian Xiaoying Prescription on intestinal mucosal barrier in rats with autoimmune thyroiditis 4.1 The results of ELISA:The serum IL-10 and colonic s Ig A levels of rats in the model group decreased,and the serum IL-10 and colonic s Ig A levels increased in the selenium yeast group and the Qijian xiaoying prescription low-dose,medium-dose,high-dose group.4.2 The results of HE staining:In the model group,the colonic mucosal epithelium was partially broken and incomplete,and the mucosal crypts were distorted with lymphocyte infiltration.After the intervention of selenium yeast and different doses of Qijian Xiaoying Prescription,the integrity of colonic mucosa of rats was restored,and the lymphocyte infiltration was significantly reduced.4.3 The results of Transmission Electron Microscope:In the model group,the tight junctions between epithelial cells of the colonic mucosa of rats were partially broken,the junctions were open and loose,the density of desmosomes decreased,the number of microvilli decreased,and the arrangement was disordered.The continuity of the tight junctions among the colonic mucosal cells of the selenium yeast group and Qijian xiaoying prescription low-dose,medium-dose,high-dose group were restored to varying degrees,the connection was tighter,the number of intestinal epithelial microvilli increased,and the arrangement was more orderly than that of the model group.4.4 The results of western-blotting: The protein expression of ZO-1 and Occludin in colon tissue of rats in model group decreased,and the protein expression of ZO-1 and Occludin in the colon tissue of the selenium yeast group and Qijian xiaoying prescription low-dose,medium-dose,high-dose group increased.Conclusion: 1.Qijian Xiaoying Prescription may regulate the expression of key proteins in the TLR4/My D88/NF-κB signaling pathway in AIT rats,reduce the production of pro-inflammatory cytokines,improve the inflammatory injury of the thyroid follicle structure,and protect the thyroid.2.Qijian Xiaoying Prescription may play a therapeutic role by adjusting the diversity and composition of the intestinal flora of AIT rats,and improving the intestinal mucosal barrier damage.