| Objective:This study aims to investigate the therapeutic effects and underlying mechanisms of Prunella vulgaris(PV)and Scutellaria barbata(SB)on autoimmune thyroiditis in rats based on the balance of CD4+T lymphocyte subsets.The results of this study could provide experimental evidence for the clinical management of autoimmune thyroiditis.Methods:Forty female Lewis rats were used in this study and divided randomly into the normal group and the AIT model group.The AIT model was established by inducing high iodine and subcutaneous injection of porcine thyroglobulin adjuvant.After six weeks,the AIT model rats were randomly divided into four groups:the model group,low-dose group(0.78 g·kg-1·d1),medium-dose group(1.56 g·kg-1·d-1),and high-dose group(3.12 g-kg-1·d-1)of PV and SB,with seven rats in each group.The rats in the three dose groups were given 0.01 mL·g-1·d-1 corresponding volume of drug to water extract suspension,while the rats in other groups were given deionized water by gavage for 8 weeks.After the intervention period,blood samples were taken from the orbital veins of rats to detect the distribution ratios of Thl,Th2,Th17,and regulatory T lymphocytes(Treg)using flow cytometry,and calculated the changes in the Th1/Th2 and Th17/Treg ratios.After collecting blood from the abdominal aorta,the concentrations of triiodothyronine(T3),free triiodothyronine(FT3),thyroxine(T4),free thyroxine(FT4),and thyroid-stimulating hormone(TSH),thyroglobulin antibody(TGAb),thyroid peroxidase antibody(TPOAb)and TSH receptor antibody(TRAb)in serum were measured using enzyme-linked immunosorbent assay(ELISA).The thyroid tissue collected from the rats was fixed in 4%paraformaldehyde,and then subjected to HE staining to observe pathological changes under a light microscope.Immunohistochemistry was also performed to analyze the protein expression levels of Interferon-γ(IFN-γ),Interleukin-4(IL-4),Interleukin17(IL-17),and Transforming growth factor beta(TGF-β)in the thyroid tissue.Results:1.Thyroid antibody detection:The serum levels of TPOAb,TGAb and TRAb in the model group significantly increased compared to the normal group(P<0.01).However,after the water extract intervention of PV and SB,the concentrations of TPOAb and TGAb in the three dose groups decreased compared to the model group(P<0.05),and the concentrations of TRAb in the high dose groups decreased(P<0.05).2.Thyroid function test:Compared with the normal group,the concentrations of T3,T4,FT3,FT4 in the model group significantly increased,while the concentration of TSH decreased(P<0.01).Compared with the model group,the concentrations of T3,T4,FT3,FT4 in the low and medium dose groups of PV and SB decreased,and the concentration of TSH increased(P<0.05).The concentrations of T3(P<0.01),T4(P<0.05),FT3(P<0.01),FT4(P<0.05)in the high dose group decreased and TSH increased(P<0.05),with significant differences observed.3.Morphological examination:The normal group showed numerous intact,tightly arranged,and appropriately sized thyroid follicles,with a rich content of colloid.The thyroid follicular epitheliums were intact.In the model group,the thyroid follicles were visibly destroyed or atrophied and the epithelial cells became thinner,with a significant reduction or absence of colloid content in the follicular lumen,and lymphocyte infiltration in the interstitium.Compared to the model group,the low-dose group of PV and SB showed a reduction in thyroid follicle destruction.The middle and high-dose groups showed relatively intact structures of the thyroid follicles and epitheliums,with a less significant reduction in colloid content in the follicular lumen and a significant decrease in lymphocyte infiltration in the interstitium compared to the model group.4.Flow cytometry analysis:Compared to the normal group,the proportion of Th1(P<0.01)and Th2(P<0.05)cells in peripheral blood of model group rats increased,with an elevated Thl/Th2 ratio but no statistically significant difference.The proportion of Th17 cells increased(P<0.05),while Treg cells decreased without significant difference,leading to an increase in the Th17/Treg ratio(P<0.01).In comparison with the model group,the PV and SB high-dose group reduced the proportions of both Th1 and Th2 cells(P<0.01),with an insignificant increase in the Th1/Th2 ratio.Additionally,the proportion of Th17 cells decreased(P<0.05),while Treg cells increased,resulting in a significant decrease in the Th17/Treg ratio(P<0.01).5.Immunohistochemistry test:Compared with the normal group,the IOD/area values of IFN-γ,IL-4,and IL-17 in the thyroid tissue of rats in the AIT model group were up-regulated,while the IOD/area value of TGF-β was down-regulated(P<0.01).Compared with the model group,the IOD/area value of IL-17 in the PV and SB low-dose group decreased(P<0.05);The IOD/area values of IL-4 and IL-17 in the medium-dose group decreased,while the IOD/area value of TGF-β increased(P<0.01);The IOD/area values of IFN-γ(P<0.05),IL-4(P<0.01),and IL-17(P<0.01)in the high-dose group decreased,while the IOD/area value of TGF-βincreased(P<0.01).All had significant differences.Conclusion:1.PV and SB have been shown to reduce the levels of thyroid autoantibodies TPOAb,TGAb and TRAb.These herbs can help regulate thyroid function and improve pathological changes in the thyroid tissue,such as damaged follicular structure and lymphocyte infiltration.2.PV and SB have not shown significant effects in correcting Thl/Th2 deviation.However,they may alleviate AIT by reducing the ratio of Thl and Th2 immune cells in peripheral blood and suppressing the expression of cytokines IFN-y and IL-4 in thyroid tissue.This helps to inhibit immune responses and reduce inflammatory damage.3.PV and SB have been shown to reduce the ratio of Th17 cells while increasing the ratio of Treg cells.They can also decrease the expression of IL-17 protein in thyroid tissue while increasing the expression level of TGF-β.This helps to correct the imbalance between Th17 and Treg cells,inhibiting inflammation and autoimmune attacks on tissue damage,thus playing a role in treating AIT. |
