Korean Red Ginseng is usually a popular herbal medicine and is

Korean Red Ginseng is usually a popular herbal medicine and is widely used in several food products. KRG may have protective effects in vascular diseases by upregulating TrxR1 in endothelial cells, thereby inhibiting the generation of reactive oxygen species and cell death. 1. Introduction The vascular endothelium is composed of a monolayer of cells and is an important organ for controlling vascular functions [1, 2]. The endothelium has multiple functions including regulation of vascular growth and remodeling, modulation of immune and thrombotic responses, and control of homeostasis and angiogenesis through interactions between the vessel wall and the immune cells [3, 4]. Impairment of endothelial function is usually implicated in most cardiovascular diseases, including diabetes, atherosclerosis, coronary artery disease, and hypertension [5, 6]. Oxidative stress-generated reactive oxygen species (ROS) are a major cause of endothelial dysfunction and damage endothelial cells by increasing the antioxidant defense mechanisms, altering vascular integrity, and IgG1 Isotype Control antibody (PE-Cy5) increasing the accumulation of lipids, and protein peroxidation [7C9]. These alterations in the structure and function of vessel cells promote vascular disease [10]. Korean Reddish Ginseng (KRG) is usually a traditional herbal medicine. Its consumption has recently been increasing owing to its inclusion in many food products, such as candy, snacks, jellies, and beverages [11]. KRG has many pharmacological and physiological protective benefits in various biological systems, 325143-98-4 supplier including anti-inflammatory, antioxidant, anticancer, and antidiabetic effects [12, 13]. Recently, a number of studies have exhibited the vascular protective properties of KRG including vasorelaxing and hypotensive effects [13, 14]. KRG increases the levels of nitric oxide (NO) and endothelial NO synthase (eNOS), which prevent endothelial cell damage and dysfunction [12, 15]. In addition, KRG protects endothelial cells by abrogating the production of NADPH-driven superoxide [14, 16], increases angiogenesis by signaling pathway activation, and enhances endothelial function [17]. These protective effects of KRG may be beneficial in cardiovascular diseases 325143-98-4 supplier including diabetes, hypertension, and atherosclerosis [18, 19]. Thioredoxin reductases (TrxR) are a family of selenocysteine-containing oxidoreductases that restore the reduced state of oxidized Thioredoxin (Trx) using electrons from NADPH + H+ [20]. You 325143-98-4 supplier will find three mammalian isoforms of TrxR: cytosolic TrxR1, mitochondrial TrxR2, and a testes-specific TrxR3 [21]. TrxR1 is well known as an antioxidant enzyme that modulates cellular function such as antiapoptosis, cell growth, and anti-inflammation [22, 23]. Thioredoxin systems, comprising Trx, TrxR, and NADPH, are highly conserved and play an essential role in redox regulation of immunomodulation, apoptosis, DNA synthesis, and cytotoxicant-induced oxidative stress [24C26]. Oxidized Trx regenerates other antioxidant enzymes such as peroxiredoxin and influences transcription factors including Fos, Jun, and p53, which decrease the levels of ROS [27]. Recent research findings suggest that suppression of the Thioredoxin system may cause oxidative stress and promote apoptotic cell death [21, 28]. In this study, we examined the role of TrxR1 in the vascular protective effects of KRG on endothelial cells. We exhibited that KRG water extract upregulated the expression of TrxR1 in human umbilical vein endothelial cells (HUVECs). We have also investigated the involvement of ROS, p38, and PKC-signaling pathways, in the induction of KRG-stimulated TrxR1 in HUVECs. 2. Materials and Methods 2.1. Materials Korean Red Ginseng powder was supplied by the Korea Ginseng and Tobacco Central Research Institute (Daejeon, Korea). M199 medium and fetal bovine serum were obtained from Welgene Inc. (Daegu, Korea). TRIzol reagent and Lipofectamine RNAiMAX were obtained from Invitrogen (Carlsbad, CA). Auranofin was purchased from Sigma Chemical (St. Louis, 325143-98-4 supplier MO). Anti-Thioredoxin reductase 1 and anti-GAPDH were purchased from AbFrontier (Seoul, Korea). SB203580 and Rottlerin were supplied by Calbiochem (La Jolla, CA), p38 siRNA (#6564) was obtained from Cell Signaling Technology (Beverly, MA), and PKC-siRNA (SC-36253) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals and reagents were of analytical grade. 2.2. Preparation of Red Ginseng Water Extract For preparation of Red Ginseng water extract, we adapted a method used in a previous study [29]. Korean Reddish Ginseng powder was soaked in water (1?:?25, w?:?w) for 3?h and then boiled for 40?min. After centrifugation at 3,000?rpm for 60?min, the supernatants of ginseng extract were further centrifuged at 13,000?rpm for 30?min and lyophilized. The resultant ginseng extracts were dissolved in pure water immediately prior to the experiment. 2.3. Cell Culture HUVECs were managed in M199 medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, 10?ng/mL human fibroblast growth factor, and 18?mU/mL heparin. The cells were produced at 37C in a 5% CO2 atmosphere. HUVECs were grown to approximately 80% confluence,.