Anti-JUN (aa 88-93) polyclonal antibody

Graves' orbitopathy (GO) is characterised in early stages by orbital fibroblast inflammation, which can be aggravated by oxidative stress and often leads to fibrosis. Protein tyrosine protein 1B (PTP1B) is a regulator of inflammation and a therapeutic target in diabetes. We investigated the role of PTP1B in the GO mechanism using orbital fibroblasts from GO and healthy non-GO subjects. After 24 hours of transfection withPTPN1siRNA, the fibroblasts were exposed to interleukin (IL)-1 beta, cigarette smoke extract (CSE), H2O2, and transforming growth factor (TGF)-beta stimulations. Inflammatory cytokines and fibrosis-related proteins were analysed using western blotting and/or enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) release was detected using an oxidant-sensitive fluorescent probe. IL-1 beta, tumor necrosis factor (TNF)-alpha, bovine thyroid stimulating hormone (bTSH), high-affinity human stimulatory monoclonal antibody of TSH receptor (M22), and insulin-like growth factor-1 (IGF-1) significantly increased PTP1B protein production in GO and non-GO fibroblasts.PTPN1silencing significantly blocked IL-1 beta-induced inflammatory cytokine production, CSE- and H2O2-induced ROS synthesis, and TGF-beta-induced expression of collagen I alpha, alpha-smooth muscle actin (SMA), and fibronectin in GO fibroblasts. SilencingPTPN1also decreased phosphorylation levels of Akt, p38, and c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER)-stress response proteins in GO cells. PTP1B may be a potential therapeutic target of anti-inflammatory, anti-oxidant and anti-fibrotic treatment of GO.

Vascular smooth muscle cell (VSMC) phenotypic switching is a hallmark of vascular remodeling that contributes to atherosclerotic diseases. MicroRNA 4463 (miR-4463) has been implicated in the development of arteriosclerosis obliterans, whereas the underlying mechanisms in VSMCs have not been fully addressed. In this study, we assessed whether miR-4463 is involved in the phenotypic switching process in VSMCs. Oxidized low-density lipoprotein (Ox-LDL, 50 mg/L) was used to simulate the oxidative stress condition, and miR-4463 expression in VSMCs was detected by a quantitative polymerase chain reaction. To determine the effect of Ox-LDL-mediated regulation of miR-4463 on the phenotypic switching of VSMCs, cell counting kit-8, cell migration assays, and cytoskeleton test were performed. After using specific antagonists of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), the relationship between miR-4463 and its downstream signaling proteins was explored. Ox-LDL induced oxidative stress to promote VSMC transformation from contraction to secretion, which clearly decreased the level of miR-4463. Then, downregulated miR-4463 enhanced the migration and phenotypic transformation of VSMCs and activated the phosphorylation of JNK and ERK; these effects were increased after Ox-LDL induction. As expected, inhibiting the two signaling proteins blocked the effect of the miR-4463 inhibitor combined with Ox-LDL. In addition, inhibition of miR-4463 led to the upregulation of basic fibroblast growth factor (bFGF) expression. The results of this study demonstrate that miR-4463 is a novel regulator of VSMC function in hypoxic conditions and modulates VSMC phenotypic switching via the JNK and ERK signaling pathways; bFGF may be the target gene of miR-4463.