Traditional Chinese medicine in thyroid-associated orbitopathy.

PURPOSE: Orbital fibroblasts (OF) are considered the central target cells in the pathogenesis of thyroid-associated orbitopathy (TAO), which comprises orbital inflammation, orbital tissue edema, adipogenesis, fibrosis, oxidative stress and autophagy. Certain active ingredients of traditional Chinese medicine (TCM) demonstrated inhibition of TAO-OF in pre-clinical studies and they could be translated into novel therapeutic strategies. METHODS: The pertinent and current literature of pre-clinical studies on TAO investigating the effects of active ingredients of TCM was reviewed using the NCBI PubMed database. RESULTS: Eleven TCM compounds demonstrated inhibition of TAO-OF in-vitro and three of them (polydatin, curcumin, and gypenosides) resulted in improvement in TAO mouse models. Tanshinone IIA reduced inflammation, oxidative stress and adipogenesis. Both resveratrol and its precursor polydatin displayed anti-oxidative and anti-adipogenic properties. Celastrol inhibited inflammation and triptolide prevented TAO-OF activation, while icariin inhibited autophagy and adipogenesis. Astragaloside IV reduced inflammation via suppressing autophagy and inhibited fat accumulation as well as collagen deposition. Curcumin displayed multiple actions, including anti-inflammatory, anti-oxidative, anti-adipogenic, anti-fibrotic and anti-angiogenic effects via multiple signaling pathways. Gypenosides reduced inflammation, oxidative stress, tissue fibrosis, as well as oxidative stress mediated autophagy and apoptosis. Dihydroartemisinin inhibited OF proliferation, inflammation, hyaluronan (HA) production, and fibrosis. Berberine attenuated inflammation, HA production, adipogenesis, and fibrosis. CONCLUSIONS: Clinical trials of different phases with adequate power and sound methodology will be warranted to evaluate the appropriate dosage, safety and efficacy of these compounds in the management of TAO.

The Role of Mitochondria-Linked Fatty-Acid Uptake-Driven Adipogenesis in Graves Orbitopathy.

CONTEXT: Depot-specific expansion of orbital adipose tissue (OAT) in Graves orbitopathy (GO; an autoimmune condition producing proptosis, visual impairment and reduced quality of life) is associated with fatty acid (FA)-uptake-driven adipogenesis in preadipocytes/fibroblasts (PFs). OBJECTIVE: This work sought a role for mitochondria in OAT adipogenesis in GO. METHODS: Confluent PFs from healthy OAT (OAT-H), OAT from GO (OAT-GO) and white adipose tissue in culture medium compared with culture medium containing a mixed hormonal cocktail as adipogenic medium (ADM), or culture-medium containing FA-supplementation, oleate:palmitate:linoleate (45:30:25%) with/without different concentration of mitochondrial biosubstrate adenosine 5'-diphosphate/guanosine 5'-diphosphate (ADP/GDP), AICAR (adenosine analogue), or inhibitor oligomycin-A for 17 days. Main outcome measures included oil-red-O staining and foci count of differentiated adipocytes for in vitro adipogenesis, flow cytometry, relative quantitative polymerase chain reaction, MTS-assay/106 cells, total cellular-ATP detection kit, and Seahorse-XFe96-Analyzer for mitochondria and oxidative-phosphorylation (OXPHOS)/glycolysis-ATP production analysis. RESULTS: During early adipogenesis before adipocyte formation (days 0, 4, and7), we observed OAT-specific cellular ATP production via mitochondrial OXPHOS in PFs both from OAT-H and OAT-GO, and substantially disrupted OXPHOS-ATP/glycolysis-ATP production in PFs from OAT-GO, for example, a 40% reduction in OXPHOS-ATP and trend-increased glycolysis-ATP production on days 4 and 7 compared with day 0, which contrasted with the stable levels in OAT-H. FA supplementation in culture-medium triggered adipogenesis in PFs both from OAT-H and OAT-GO, which was substantially enhanced by 1-mM GDP reaching 7% to 18% of ADM adipogenesis. The FA-uptake-driven adipogenesis was diminished by oligomycin-A but unaffected by treatment with ADP or AICAR. Furthermore, we observed a significant positive correlation between FA-uptake-driven adipogenesis by GDP and the ratios of OXPHOS-ATP/glycolysis-ATP through adipogenesis of PFs from OAT-GO. CONCLUSION: Our study confirmed that FA uptake can drive OAT adipogenesis and revealed a fundamental role for mitochondria-OXPHOS in GO development, which provides potential for therapeutic interventions.

A case report of thyroid-associated Orbitopathy with elevated TPO antibodies.

BACKGROUND: Thyroid associated orbitopathy (TAO) is defined as an immune mediated inflammatory process affecting the extraocular muscles, connective and adipose tissue of uncertain etiopathogenesis. TAO are classically described in Grave's disease (GD) however it may occur in euthyroid and hypothyroid patients. Those patients usually test positive for Thyroid Stimulating Hormone receptor antibodies (TRAb). For instance, only few cases of severe Hashimoto's thyroiditis (HT) associated orbitopathy with negative TRAb are reported to date. CASE PRESENTATION: Herewith we report a rare case of a middle-aged female who presented with bilateral progressive upper and lower palpebral edema and a unilateral marked proptosis associated with asthenia, headache and decrease in visual acuity. Biological investigation was notable for high levels of anti-thyroid peroxidase antibodies (Anti-TPO) in an otherwise euthyroid patient with negative TRAb. Orbital Magnetic resonance imaging revealed edema of the extraocular muscles and inflammation of periorbital soft tissue. The patient received a treatment with intravenous methylprednisolone followed by oral treatment with prednisone. This regimen was both effective and safe with minimal metabolic side effects in our patient. CONCLUSION: Minor ocular manifestations of HT are common; however, severe sight threatening ophtalmopathy in the absence of TRAb is rare. Multiple differential diagnosis should be considered and investigated before diagnosing this rare entity. Management of similar cases is currently based on reports and no clear guidelines have been elaborated, corticosteroids is the mainstream of treatment with a potential benefit of selenium supplementation in mild to moderate cases.

Gypenosides Protect Orbital Fibroblasts in Graves Ophthalmopathy via Anti-Inflammation and Anti-Fibrosis Effects.

Purpose: To investigate the effect of Gypenosides (Gyps) on the inflammation and fibrosis in orbital fibroblasts (OFs) in Graves ophthalmopathy (GO). Methods: Bioinformatics analyses were performed to identify the enriched genes and signaling pathways related to Gyps function. For ex vivo experiments, OFs were cultured from orbital connective tissues from patients with GO. OF proliferation was estimated by Cell Counting Kit-8 assay. Effects of Gyps treatment on interleukin (IL)-1ß-induced inflammation and transforming growth factor-ß1 (TGF-ß1)-induced fibrosis were evaluated by real-time quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting. OFs were treated with IL-1ß or TGF-ß1 in the absence or presence of Gyps pretreatment, and the levels of related mRNA or proteins were evaluated by RT-qPCR or ELISA. Results: Eight inflammation-related target genes and nine fibrosis-related target genes were screened out. These genes were mainly enriched in pathways corresponding to inflammation and fibrosis, respectively. IL-1ß-induced upregulation of inflammatory cytokines, and TGF-ß-induced upregulation of fibrotic mediators in OFs were downregulated by Gyps. Moreover, Gyps reduced the activation of Toll like receptors 4/nuclear factor-κ B signaling and TGF-ß1/SMAD2/SMAD4 signaling in GO OFs. Conclusions: Gyps could protect GO-derived OFs against IL-1ß-induced inflammation and TGF-ß1-induced fibrosis. Thus Gyps might have therapeutic potential on inflammation and fibrosis in GO.

Therapeutic Effect of Protocatechuic Aldehyde in an In Vitro Model of Graves' Orbitopathy.

PURPOSE: Protocatechuic aldehyde (3,4-dihydroxybenzaldehyde; PCA) is extracted from Salvia miltiorrhiza, and has been reported to possess antiproliferative, antioxidant, and antiadipogenesis properties in various in vivo and in vitro experiments. This study aimed to outline the antioxidant and suppressive effects of PCA on adipogenesis and hyaluronan production in orbital fibroblasts to help with designing therapeutic approaches for Graves' orbitopathy (GO). METHODS: We assessed the in vitro effects of PCA on orbital fibroblasts, which were cultured from orbital fat tissue obtained from patients undergoing orbital decompression for severe GO. Control tissue was obtained from patients undergoing orbital surgery with no history of GO or Graves' hyperthyroidism. RESULTS: The 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt assay results confirmed the free radical scavenging effect of PCA after treatment. Protocatechuic aldehyde exhibited a suppressive effect on intracellular reactive oxygen species generation and upregulated heme oxygenase-1 expression in Western blot analysis. Protocatechuic aldehyde attenuated TNF-α and IL-1ß-induced hyaluronan production. Oil Red-O staining results revealed a decrease in lipid droplets and suppressed expression of the adipogenesis-related proteins peroxisome proliferator-activated receptor (PPAR)-γ, CCAAT/enhancer binding protein (c/EBP)-α, and c/EBP-ß upon treatment with PCA during adipose differentiation. CONCLUSIONS: In this study, PCA exerted significant antioxidant and antiadipogenic effects and inhibited the production of hyaluronan in GO orbital fibroblasts. Accordingly, PCA potentially could be used as a novel treatment option for GO.

Development of a Novel Thyroid Function Fluctuated Animal Model for Thyroid-Associated Ophthalmopathy.

BACKGROUND: The establishment of a suitable and stable animal model is critical for research on thyroid-associated ophthalmopathy (TAO). In clinical practice, we found that patients treated with I-131 often exhibit TAO; therefore, we aimed to establish a novel thyroid function fluctuated animal model of TAO by simulating the clinical treatment process. METHODS: We treated SD rats with I-131 to damage the thyroid and then used sodium levothyroxine (L-T4) to supplement the thyroid hormone (TH) levels every seven days, leading to a fluctuating level of thyroid hormones that simulated the status of clinical TAO patients. Rats administered normal saline were considered as a control. The weight, intraocular pressure, and serum T3, T4, TSH and TRAb levels of the rats were measured, and the pathological changes were analyzed by H&E staining and transmission electron microscopy (TEM). RESULTS: The experimental rats (TAO group) exhibited significantly reduced weight and elevated intraocular pressure compared with the control rats. Meanwhile, the serum levels of T3 and T4 were up-regulated in the TAO group, but the TSH level decreased during the 10-week study. Moreover, increased numbers of blood vessels and inflammatory cell infiltrations were observed in the orbital tissues of the TAO rats, while no abnormal changes occurred in the control rats. The orbital myofibrils in the TAO rats appeared fractured and dissolved, with twisted structures. Mitochondrial swelling and vacuoles within the endoplasmic reticulum, swelling nerve fibers, shedding nerve myelin, and macrophages were found in the TAO group. CONCLUSION: Rats treated with I-131 and sodium levothyroxine exhibited characteristics similar to those of TAO patients in the clinic, providing an effective and simple method for the establishment of a stable animal model for research on the pathogenesis and treatment of TAO.

The tyrosine kinase inhibitor dasatinib effectively blocks PDGF-induced orbital fibroblast activation.

BACKGROUND: Graves' ophthalmopathy (GO) remains hard to treat. Excessive orbital fibroblast activation by platelet-derived growth factor (PDGF)-BB contributes to GO. The tyrosine kinase inhibitors (TKIs) imatinib mesylate and dasatinib both target PDGF-receptor tyrosine kinase activity, albeit with a different potency. We compared the efficacy of these TKIs on PDGF-BB-induced proliferation, and on cytokine and hyaluronan production by orbital fibroblasts. Also the capacity of dasatinib to suppress GO-associated gene expression in orbital tissue was examined. METHODS: Orbital fibroblasts from four GO patients and five control subjects were used. The efficacy of the two TKIs was tested by: 1) pre-incubating orbital fibroblasts overnight with different TKI concentrations, followed by 24 h stimulation with PDGF-BB, 2) adding TKI and PDGF-BB simultaneously to the orbital fibroblasts in 24 h cultures. Proliferation was assessed by colorimetric assay. Hyaluronan and cytokine production were measured by ELISA. Furthermore, orbital tissue was obtained from a patient with active GO, and the effect of dasatinib on the expression levels of HAS2-, CCL2-, IL6-, and IL8-mRNA expression was examined by real-time quantitative PCR. RESULTS: Pre-incubation of orbital fibroblasts with imatinib mesylate or dasatinib resulted in significant and dose-dependent inhibition of PDGF-BB-induced orbital fibroblast proliferation, and hyaluronan and cytokine production. Dasatinib exhibited these effects at far lower concentrations. The same results were observed in the setting where TKI and PDGF-BB treatments were commenced simultaneously. In orbital tissue from active GO, dasatinib significantly suppressed HAS2-, CCL2-, IL6- and IL8-mRNA levels. CONCLUSION: Dasatinib may be a promising alternative to high-dose steroids in the treatment of GO.

Pingmu decoction enhances apoptosis of orbital adipocytes derived from patients with Graves' ophthalmophathy.

Graves' ophthalmopathy (GO), an autoimmune disease, has been demonstrated to result from an increased volume of orbital contents, including adipose, connective and extraocular muscle tissues. In our previous study, we showed that Pingmu decoction is capable of alleviating GO progression. In this study, to further investigate the underlying mechanism(s), we examined the effects of Pingmu decoction-containing serum on the proliferation and apoptosis of preadipocytes and adipocytes derived from the orbital adipose tissue of GO patients. Our data demonstrate for the first time that Pingmu decoction-containing serum significantly reduces preadipocyte proliferation and increases adipocyte apoptosis as measured by MTT assay and Annexin V/FITC staining, respectively. Moreover, Pingmu decoction elevated the levels of cleaved caspase-3, 8 and 9 and decreased the levels of cell cycle-related genes compared to those of the control. In addition, cell cycle arrest at the G0/G1 phase was observed following Pingmu decoction treatment. This study shows a mechanism by which Pingmu decoction serves as an effective GO medicine by downregulating preadipocyte proliferation and increasing adipocyte apoptosis.

Inhibitory effects of Triptolide on interferon-gamma-induced human leucocyte antigen-DR, intercellular adhesion molecule-1, CD40 expression on retro-ocular fibroblasts derived from patients with Graves' ophthalmopathy.

PURPOSE: To explore the effects of Triptolide, the principal active diterpenoid from the Chinese Medicinal Herb Tripterygium Wilfordii Hook F that has immunosuppressive and anti-inflammatory properties, on cell proliferation, hyaluronic acid (HA) synthesis, and the expressions of human leucocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) and CD40 on cultured retro-ocular fibroblasts (RFs) from patients with Graves' ophthalmopathy. METHODS: After two to five passages, cultured RFs were incubated for 48 h within a medium alone or in the presence of recombinant human interferon-gamma (IFN-gamma) and various concentrations of Triptolide. Cell viability was assessed by MTT (3-[4.5-dimethylahiazol-2-yl]-2,5-diphenyltetrazolium Bromide). RFs proliferation was assessed by [(3)H]-thymidine incorporation assay. Flow cytometry was used to investigate the amount of HLA-DR, ICAM-1 and CD40. HA synthesis was measured by radioimmunoassay. RESULTS: Cell viability was not detrimentally affected when incubated with Triptolide from 0.01 microg/L to 10 microg/L for 48 h, and decreased with 20 microg/L Triptolide. The incorporation of [(3)H]-thymidine of RFs was 55 476 +/- 15 842 cpm incubated with medium alone or 18 352 +/- 3568 cpm with 10 microg/L Triptolide (t = 5.600, P < 0.01). Initially, the percentage of positive cells of HLA-DR, ICAM-1 and CD40 on RFs were 4.75 +/- 2.13%, 17.53 +/- 10.12% and 6.38 +/- 2.23%, respectively, and the synthesis of HA was 100 +/- 12%. Compared with basal values, 48-h incubation with IFN-gamma (100 U/mL) significantly enhanced the amount of HLA-DR, ICAM-1 and CD40, and HA synthesis. The values were 60.58 +/- 10.12% (t = 13.224, P < 0.01), 62.66 +/- 18.17% (t = 5.315, P < 0.01), 57.67 +/- 13.61% (t = 9.110, P < 0.01) and 164 +/- 22% (t = 9.238, P < 0.01), respectively. Triptolide 0.01 microg/L had little effect on IFN-gamma-induced HLA-DR, ICAM-1 and CD40 amounts, as well as HA synthesis. When the concentration ranged from 0.1 microg/L to 10 microg/L, Triptolide inhibited IFN-gamma-induced RFs activation in a dose-dependent manner. It was also found that Triptolide had the same inhibiting effects on IFN-gamma-induced RFs and skin fibroblasts from patients with normal individual conditions. CONCLUSIONS: Triptolide could inhibit IFN-gamma-induced activation of RFs derived from patients with Graves' ophthalmopathy.