Serum Selenium Levels in Patients with Graves Disease: Associations with Clinical Activity and Severity in a Retrospective Case-control Study.

PURPOSE: To compare serum selenium levels in Graves patients and non-Graves control participants and to evaluate associations between serum selenium levels and clinical features of Graves orbitopathy (GO). METHODS: We conducted a single-center, retrospective case-control study among 33 patients with Graves disease without GO (GD), 31 patients with diagnosed GO, and 27 unaffected healthy participants enrolled between 2013 and 2020 at Severance Hospital. We compared serum selenium concentrations between the GD, GO, and healthy control groups, and analyzed associations between serum selenium and GO patients' clinical activity scores, severity (assessed through modified NOSPECS scores), and other clinical features using multivariate linear regression analysis. RESULTS: Mean serum selenium levels were 109.30 ± 16.39, 111.39 ± 14.04, and 126.09 ± 21.09 ng/mL in GO patients, GD patients, and healthy control participants, respectively. Mean serum selenium levels in Graves patients with and without orbitopathy were significantly lower than those in the healthy control group (p < 0.05), and mean selenium levels were slightly lower in GO than those in GD patients (p = 0.594). Serum selenium levels were significantly lower in GO patients with eyelid retraction than in patients without retraction (p = 0.038). However, serum selenium levels were not associated with clinical activity scores and modified NOSPECS scores (p = 0.241 and 0.801, respectively). CONCLUSIONS: Serum selenium levels were significantly lower in Graves patients with or without GO, compared to non-Graves control participants. Selenium levels were not associated with clinical activity scores or NOSPECS scores, though we observed an association with eyelid retraction.

Danshen Extracts Prevents Obesity and Activates Mitochondrial Function in Brown Adipose Tissue.

BACKGROUND: Danshen has been widely used in oriental medicine to improve body function. The purpose of this study is to investigate the effect of water-soluble Danshen extract (DE) on weight loss and on activation proteins involved in mitochondrial biogenesis in brown adipose tissue (BAT) in obese mice. METHODS: BAT was isolated from 7-week-old male Sprague-Dawley rats, and expression of proteins related to mitochondrial biogenesis was confirmed in both brown preadipocytes and mature brown adipocytes treated with DE. For the in vivo study, low-density lipoprotein receptor knock out mice were divided into three groups and treated for 17 weeks with: standard diet; high fat diet (HFD); HFD+DE. Body weight was measured every week, and oral glucose tolerance test was performed after DE treatment in streptozotocin-induced diabetic mice. To observe the changes in markers related to thermogenesis and adipogenesis in the BAT, white adipose tissue (WAT) and liver of experimental animals, tissues were removed and immediately frozen in liquid nitrogen. RESULTS: DE increased the expression of uncoupling protein 1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha in brown preadipocytes, and also promoted the brown adipocyte differentiation and mitochondrial function in the mature brown adipocytes. Reactive oxygen species production in brown preadipocytes was increased depending on the concentration of DE. DE activates thermogenesis in BAT and normalizes increased body weight and adipogenesis in the liver due to HFD. Browning of WAT was increased in WAT of DE treatment group. CONCLUSION: DE protects against obesity and activates mitochondrial function in BAT.

Therapeutic Effect of Guggulsterone in Primary Cultured Orbital Fibroblasts Obtained From Patients with Graves' Orbitopathy.

Purpose: Inflammation, hyaluronan production, and adipogenesis are the main pathological events leading to Graves' orbitopathy (GO). Guggulsterone (GS), a phytosterol found in the resin of the guggul plant, is a well-known treatment for several inflammatory disorders, such as arthritis, obesity, and hyperlipidemia. Here we investigated the effects of GS treatment on GO pathology. Methods: Using primary cultures of orbital fibroblasts from GO patients and non-GO controls, we examined the effects of GS on hyaluronan production and the production of proinflammatory cytokines induced by interleukin (IL)-1ß, using real-time reverse transcription-polymerase chain reaction analysis, western blots, and enzyme-linked immunosorbent assays. Further, adipogenic differentiation was evaluated by quantification of Oil Red O staining and assessment of protein levels of peroxisome proliferator activator gamma (PPARγ), CCAAT-enhancer-binding proteins (C/EBP) α and ß, and sterol regulatory element-binding protein-1 (SREBP-1). Results: Treatment with noncytotoxic concentrations of GS resulted in the dose-dependent inhibition of IL-1ß-induced inflammatory cytokines, including IL-6, IL-8, MCP-1, and COX-2, at both mRNA and protein levels. The hyaluronan level was also significantly suppressed by GS. Moreover, GS significantly decreased the formation of lipid droplets and expression of PPARγ, C/EBP α/ß, and SREBP-1 in a dose-dependent manner. GS pretreatment attenuated the phosphorylation of nuclear factor-kappa B induced by IL-1ß. Conclusions: Our data show significant inhibitory effects of GS on inflammation, production of hyaluronan, and adipogenesis in orbital fibroblasts. To our knowledge, this is the first in vitro preclinical evidence of the therapeutic effect of GS in GO.

Therapeutic Effect of Curcumin, a Plant Polyphenol Extracted From Curcuma longae, in Fibroblasts From Patients With Graves' Orbitopathy.

Purpose: We examined the therapeutic effect of nontoxic concentrations of curcumin, a plant polyphenol extracted from Curcuma longae, in primary cultures of orbital fibroblasts from Graves' orbitopathy (GO). Methods: The effect of curcumin on interleukin (IL)-1ß induced-proinflammatory cytokine production was determined using quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. Adipogenic differentiation was identified using Oil-Red O staining, and levels of peroxisome proliferator activator γ (PPARγ) and CCAAT-enhancer-binding proteins (C/EBP) α/ß were determined by Western blot analyses. Antioxidant activity was measured using an oxidant-sensitive fluorescent probe to detect intracellular reactive oxygen species (ROS) generated in response to hydrogen peroxide (H2O2) and cigarette smoke extract (CSE). Results: Treatment with curcumin resulted in a dose- and time-dependent decrease in IL-1ß-induced synthesis of inflammatory cytokines, including IL-6, IL-8, MCP-1, and ICAM-1 at both mRNA and protein levels. Decrease in lipid droplets and expression of PPARγ and c/EBPα/ß were noted in fibroblasts treated with curcumin during adipose differentiation. CSE- or H2O2-induced ROS synthesis was significantly lower in curcumin-treated fibroblasts in comparison with the control. Curcumin significantly suppressed IL-1ß-induced phosphorylated extracellular signal-regulated kinase, Akt, c-Jun NH(2)-terminal kinase, and nuclear factor κ-light-chain-enhancer of activated B cells, p65 proteins and stimulated ß-catenin translocation into nucleus during adipogenesis. Conclusions: Curcumin inhibits proinflammatory cytokine production, ROS synthesis, and adipogenesis in orbital fibroblasts of GO patients in vitro possibly related to multiple proinflammatory signaling molecules and ß-catenin pathway. The results of the study support potential use of the curcumin in the treatment of 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.

Inhibitory Effects of α-Lipoic Acid on Oxidative Stress-Induced Adipogenesis in Orbital Fibroblasts From Patients With Graves Ophthalmopathy.

A choice of the optimal treatment for Graves ophthalmopathy (GO) is a challenge due to the complexity of the pathogenesis. Alpha-lipoic acid (ALA) is well known as a multifunctional antioxidant, helping to protect cells against oxidative stress and inflammatory damage.The aim of this study was to investigate the effects of ALA on intracellular production of reactive oxygen species (ROS), inflammation, and adipogenesis using primary cultured orbital fibroblasts from patients with GO.Intracellular ROS levels and mRNA expressions of proinflammatory cytokines and chemokines including intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and regulated upon activation normal T cell expressed and presumably secreted (RANTES) were measured. After adipogenesis, the expressions of peroxisome proliferator-activated receptor (PPAR)γ, CCAAT-enhancer-binding proteins (C/EBP)α and ß, and heme oxygenase-1 (HO-1) were investigated.H2O2 dose-dependently stimulated ROS production and HO-1 expression. Addition of ALA strongly attenuated ROS production and further increased HO-1 expression. However, by pretreatment of zinc protoporphyrin (ZnPP), HO-1 inhibitor, ALA inhibition of ROS generation by H2O2 was abolished. Tumor necrosis factor (TNF)α-induced mRNA expressions of ICAM-1, IL-6, MCP-1, and RANTES were inhibited by ALA treatment. In this context, TNFα-induced phosphorylation of P65 was also inhibited. In addition, ALA dose-dependently inhibited H2O2-induced intracellular accumulation of lipid droplets. The expression of adipogenic transcription factors, including PPARγ, C/EBPα, and ß, was also inhibited.ALA is a potential therapeutic agent for GO because of the inhibitory effects on ROS production and gene expression of proinflammatory cytokines and chemokines, resulting in prevention of adipose-tissue expansion.

Therapeutic effects of water soluble danshen extracts on atherosclerosis.

Danshen is a traditional Chinese medicine with many beneficial effects on cardiovascular diseases. The aim of this study was to evaluate the mechanisms responsible for the antiatherogenic effect of water soluble Danshen extracts (DEs). Rat vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) were treated with DE. To evaluate the effects of DE in vivo, carotid balloon injury and tail vein thrombosis were induced in Sprague-Dawley (SD) rats and iliac artery stent was induced in New Zealand white rabbits. The inhibitory action of DE on platelet aggregation was confirmed with an impedance aggregometer. DE inhibited the production of reactive oxygen species, and the migration and proliferation of platelet-derived growth factor-BB stimulated VSMCs. Furthermore, DE prevented inflammation and apoptosis in HUVECs. Both effects of DE were reconfirmed in both rat models. DE treatment attenuated platelet aggregation in both in vivo and ex vivo conditions. Pretreatment with DE prevented tail vein thrombosis, which is normally induced by κ-carrageenan injection. Lastly, DE-treated rabbits showed decreased in-stent restenosis of stented iliac arteries. These results suggest that water soluble DE modulates key atherogenic events in VSMCs, endothelial cells, and platelets in both in vitro and in vivo conditions.

Protocatechuic aldehyde inhibits migration and proliferation of vascular smooth muscle cells and intravascular thrombosis.

The migration and proliferation of vascular smooth muscle cells (VSMCs) and formation of intravascular thrombosis play crucial roles in the development of atherosclerotic lesions. This study examined the effects of protocatechuic aldehyde (PCA), a compound isolated from the aqueous extract of the root of Salvia miltiorrhiza, an herb used in traditional Chinese medicine to treat a variety of vascular diseases, on the migration and proliferation of VSMCs and platelets due to platelet-derived growth factor (PDGF). DNA 5-bromo-2'-deoxy-uridine (BrdU) incorporation and wound-healing assays indicated that PCA significantly attenuated PDGF-induced proliferation and migration of VSMCs at a pharmacologically relevant concentration (100 µM). On a molecular level, we observed down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathways, both of which regulate key enzymes associated with migration and proliferation. We also found that PCA induced S-phase arrest of the VSMC cell cycle and suppressed cyclin D2 expression. In addition, PCA inhibited PDGF-BB-stimulated reactive oxygen species production in VSMCs, indicating that PCA's antioxidant properties may contribute to its suppression of PDGF-induced migration and proliferation in VSMCs. Finally, PCA exhibited an anti-thrombotic effect related to its inhibition of platelet aggregation, confirmed with an aggregometer. Together, these findings suggest a potential therapeutic role of PCA in the treatment of atherosclerosis and angioplasty-induced vascular restenosis.

Effect of teriparatide on pregnancy and lactation-associated osteoporosis with multiple vertebral fractures.

Pregnancy and lactation-associated osteoporosis (PLO) is very rare, but it can cause severe vertebral compression fractures with disabling back pain. PLO patients have commonly been treated with antiresorptive agents against high bone turnover. There are, however, some concerns regarding the use of bisphosphonates: (1) PLO occurs during the first pregnancy with a high possibility of recurrence during the second pregnancy, (2) long-term outcomes of bisphosphonates in PLO are lacking, and (3) there is a possibility of bisphosphonates accumulated in the bones crossing the placenta. Therefore, alternative therapies must be considered. We analyzed the effect of teriparatide (TPTD), the human recombinant parathyroid hormone (1-34), for 18 months in three women with PLO. Multiple vertebral fractures with severe back pain appeared within 6 months after their first childbirth. Two of them had a family history of osteoporosis. Lactation was discontinued immediately after diagnosis of PLO. Calcium carbonate, cholecalciferol, and TPTD were prescribed. The back pain immediately resolved. Bone mineral density (BMD) increased by 14.5-25.0% (mean 19.5%) at the lumbar spine and by 9.5-16.7% (mean 13.1%) at the femoral neck, after 18 months of treatment. The final Z scores in these PLO patients were nearly normalized. Two women had a second baby without any complication. BMD significantly improved after 18 months of treatment with TPTD without further fractures. In conclusion, TPTD should be considered to avoid long-term morbidity in young patients with PLO and is highly encouraged for use in PLO patients with multiple vertebral fractures.

Natural therapeutic magnesium lithospermate B potently protects the endothelium from hyperglycaemia-induced dysfunction.

AIMS: We have investigated the effects of magnesium lithospermate B (MLB), the active compound of the Oriental herbal remedy, Salvia miltiorrhizae, on endothelial dysfunction associated with diabetes mellitus using cultured endothelial cells and an animal model of type 2 diabetes mellitus. METHODS AND RESULTS: The effect of MLB on vasodilatory function in Otsuka Long-Evans Tokushima Fatty (OLETF) rats was assessed. MLB treatment for 20 weeks starting at 12 weeks attenuated the decrease in endothelium-dependent vasodilation in OLETF rats. MLB treatment also increased serum nitrite level and reduced serum advanced glycation end products concentration. The effect of MLB was greater than an equivalent dose of alpha-lipoic acid (alphaLA), a popular antioxidant treatment. MLB rescued the inhibition of endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation in endothelial cells cultured in hyperglycaemia. This effect was dependent on Akt phosphorylation and associated with decreased O-linked N-acetylglucosamine protein modification of eNOS. MLB also increased nuclear factor erythroid 2-related factor-2 (Nrf-2) activation in a phosphoinositide 3-kinase/Akt pathway dependent manner. MLB treatment induced the expression of the Nrf-2-regulated antioxidant enzyme, heme oxygenase-1. The antioxidant alphaLA could not produce this effect. Moreover, MLB decreased oxidative stress and endothelial cell apoptosis caused by hyperglycaemia. CONCLUSION: MLB is a naturally occurring, new generation antioxidant that activates eNOS and ameliorates endothelial dysfunction in diabetes by enhancing vasodilation in addition to reducing oxidative stress. The relative strong performance of MLB makes it an ideal candidate for further, expanded trials as a new generation of antioxidant to treat diabetes-related complications.

Protective effects of magnesium lithospermate B against diabetic atherosclerosis via Nrf2-ARE-NQO1 transcriptional pathway.

Hyperglycemia-induced oxidative stress is known to play an important role in the development of several diabetic complications, including atherosclerosis. Although a number of antioxidants are available, none have been found to be suitable for regulating the oxidative stress response and enhancing antioxidative defense mechanisms. In this study, we evaluated the effects of magnesium lithospermate B (LAB) against oxidative stress. We also endeavored to identify the target molecule of LAB in vascular smooth muscle cells (VSMCs) and the underlying biochemical pathways related to diabetic atherosclerosis. Modified MTT and transwell assays showed that the increased proliferation and migration of rat aortic VSMCs in culture with high glucose was significantly inhibited by LAB. LAB also attenuated neointimal hyperplasia after balloon catheter injury in diabetic rat carotid arteries. To determine molecular targets of LAB, we studied the effects of LAB on aldose reductase (AR) activity, O-GlcNAcylation, and protein kinase C (PKC) activity in VSMCs under normoglycemic or hyperglycemic conditions and showed the improvement of major biochemical pathways by LAB. Potential involvement of the nuclear factor erythroid 2-related factor-2 (Nrf2)--antioxidant responsive element (ARE)-NAD(P)H: quinone oxidoreductase-1 (NQO1) pathway was assessed using siRNA methods. We found that LAB activates the NQO1 via the Nrf2-ARE pathway, which plays an important role in inhibition of the major molecular mechanisms that lead to vascular damage and the proliferation and migration of VSMCs. Together, these findings demonstrate that the induction of the Nrf2-ARE-NQO1 pathway by LAB could be a new therapeutic strategy to prevent diabetic atherosclerosis.

Estrogen regulates a tissue-specific calpain in the anterior pituitary.

A PCR-based cDNA subtraction hybridization was performed to identify the genes stimulated by estrogen in the pituitary. A novel tissue-specific calpain (nCL-2'), previously shown to be expressed mainly in the stomach, was markedly induced in the pituitary after estrogen treatment. The 5'-flanking region of the calpain nCL-2' gene was analyzed to assess the molecular mechanism of estrogen regulation. Sequence analysis of the nCL-2' promoter (1.9 kb) revealed a perfectly palindromic putative estrogen-response element (ERE), GGTCATGCTGACC. In transient transfection studies, the nCL-2' promoter was highly responsive to estrogen in the presence of estrogen receptor (ER). Transcriptional activation by estrogen was prevented by an ERE mutation as well as by mutations in the ER DNA-binding domain. An ER antagonist, ICI 182780, blocked estrogen inducibility of the nCL-2' promoter. We conclude that the nCL-2' form of calpain is expressed in the pituitary and upregulated by estrogen at the transcription level.