Prunella vulgaris L. Attenuates Experimental Autoimmune Thyroiditis by Inhibiting HMGB1/TLR9 Signaling.

BACKGROUND: Prunella vulgaris L. (PV) has been used to treat autoimmune thyroiditis (AIT), but the underlying mechanism remains unknown. The present study was designed to evaluate the effect of PV on AIT and explore the role of high-mobility group box-1 (HMGB1) signaling in PV-mediated effects in vivo and in vitro. METHODS: In the present study, bioactive components of PV were identified using UPLC-ESI-MS. The protective effects and potential mechanisms critical for the anti-inflammatory and immunomodulatory effects of PV in AIT were investigated in a rat model of thyroglobulin-induced experimental autoimmune thyroiditis (EAT) and in lipopolysaccharide (LPS)-induced thyroid follicular cells (TFCs). RESULTS: The main bioactive compound identified in PV was rosmarinic acid. The thyroid volume, thyroiditis inflammation score and serum thyroglobulin antibody levels of EAT rats were attenuated by PV treatment (P<0.01). In addition, PV significantly reduced the elevated levels of the proinflammatory cytokines TNF-α, IL-6, IL-1ß and monocyte chemoattractant protein-1 (MCP-1) both in vivo (P<0.01) and in vitro (P<0.05). PV downregulated HMGB1 mRNA and protein expression, reduced HMGB1 secretion, and inhibited TLR9 signaling pathways (TLR9 and MyD88) in PV-treated EAT rats and TFCs. Moreover, PV reversed the increases in the numbers of splenic Th1, Th2, and Th17 cells. Finally, our results acquired following administration of ethyl pyruvate, an HMGB1 inhibitor, to splenocytes cultured in vitro supported the hypothesis that the HMGB1/TLR9 pathway is involved in the PV-mediated reductions in Th1, Th2 and Th17 cells. CONCLUSION: PV decreased the activity of the TLR9/MyD88 pathway and proinflammatory cytokines through HMGB1. In addition, we are the first to show that PV attenuated the HMGB1-induced increases in Th1, Th2 and Th17 cells in AIT models. These findings provide new evidence for the potential therapeutic value of PV as a treatment for AIT and other autoimmune diseases.

Prunella vulgaris L. attenuates experimental autoimmune thyroiditis by inducing indoleamine 2,3-dioxygenase 1 expression and regulatory T cell expansion.

BACKGROUND: Prunella vulgaris L. (P. vulgaris) has traditionally been used to treat swelling and inflammation of the thyroid gland. This study aimed to evaluate the effects of P. vulgaris on experimental autoimmune thyroiditis (EAT) and explore the roles of indoleamine 2,3-dioxygenase 1 (IDO1) and regulatory T cells (Tregs) in these P. vulgaris-mediated effects. METHODS: The main bioactive compounds in P. vulgaris were analysed by high-performance liquid chromatography. An EAT model was established by immunization of Lewis rats with thyroglobulin via subcutaneous injection. Thyroid volume was assessed by ultrasound, and lymphatic infiltration in the thyroid was evaluated by haematoxylin and eosin staining. The serum levels of thyroglobulin antibody (TgAb) and cytokines were measured by indirect enzyme-linked immunosorbent assay. The percentage of CD4+CD25+Foxp3+ Tregs was detected by flow cytometry. The mRNA and protein levels of IDO1 were measured by qRT-PCR and Western blotting, respectively. The levels of tryptophan (Trp) and kynurenine (Kyn) in serum and faecal samples were assessed with a fluorometric kit and spectrophotometry. RESULTS: The main bioactive compound in P. vulgaris was rosmarinic acid. The TgAb level and thyroid volume in EAT rats were significantly decreased after administration of P. vulgaris (P < 0.01). The inflammation score in EAT rats that were administered P. vulgaris was significantly lower than that in the EAT controls (P < 0.01). In addition, P. vulgaris promoted the expansion of splenic Tregs and increased the production of IL-10 and TGF-ß (P < 0.01) in EAT rats. Moreover, P. vulgaris induced IDO1 mRNA and protein expression in the spleen and intestine in P. vulgaris-treated EAT rats (P < 0.01). Finally, Trp levels were reduced and Kyn levels and the Kyn/Trp ratio were increased in the serum of P. vulgaris-treated EAT rats. CONCLUSION: We were the first to demonstrate the role of IDO1-induced Treg expansion in P. vulgaris-mediated attenuation of EAT. Our study provides insight into the immunopathogenesis of autoimmune thyroiditis and shows the potential therapeutic value of P. vulgaris.

Iodine excess did not affect the global DNA methylation status and DNA methyltransferase expression in T and B lymphocytes from NOD.H-2h4 and Kunming mice.

Dysregulated DNA methylation in lymphocytes has been linked to various autoimmune disorders. Excessive iodine intake leads to lymphocyte dysfunction and contributes to autoimmune thyroiditis (AIT) flares in humans and animals. However, whether excessive iodine modifies the DNA methylation status in lymphocytes is unknown. Twenty NOD.H-2h4 mice and 20 Kunming mice were randomly divided into high iodine and control groups. We scored lymphatic infiltration in the thyroid by hematoxylin and eosin (H&E) staining and assayed serum thyroglobulin antibody (TgAb) levels by an indirect enzyme-linked immunosorbent assay. CD3+ T cells and CD19+ B cells were separated by flow cytometry. Global DNA methylation levels were examined by absorptiometry. Methylation of long interspersed nucleotide element-1 (LINE-1) repeats was detected with bisulfite sequencing PCR. Expression of DNA methyltransferase (DNMT) 1, DNMT3a and DNMT3b mRNA and protein were determined by real-time PCR and Western blot, respectively. We observed evident thyroiditis in the high­iodine-treated NOD.H-2h4 mice, while mice in the other three groups did not develop thyroiditis. No differences were found in the global methylation levels and methylation status of LINE-1 repeats in T and B lymphocytes from high­iodine-treated NOD.H-2h4 mice and Kunming mice compared with those from normal­iodine-supplemented controls. We did not find obvious changes in DNMT mRNA and protein expression levels in T and B lymphocytes among the studied groups. In conclusion, we showed for the first time that excess iodine did not affect the global methylation status or DNMT expression in T and B lymphocytes in NOD.H-2h4 and Kunming mice.