Study Of The Mechanism Of Biologic Effects By Two GPX Mimics

Oxidative Stress is a kind of unbalance state between oxidation and antioxidation. Reactive oxidative species (ROS) are harmful to cells when oxidative metabolism products increase or the antioxidative defenses lack in the organism. ROS have highly activity and very strong oxidative ability and they can attack the molecules by the oxidation, and then make the big molecules denaturation, cross-linking and breakout, and then lead to the damage of cell structure and function and the injury of the tissue.Lots of researches showed that reactive oxygen species (ROS) play a pivotal role in the pathogenesis of Parkinson's Disease and atherosclerosis. Treatment with antioxidants may prevent or reduce the rate of progression of these diseases by elimination of Ros, enhancement of the antioxidant defence and increase of antioxidative capacity.Glutathione peroxidase (GPX) is a member of the antioxidant enzyme system, which plays an important role in protection of organism. GPX prevent the organism from the damage of free radicals by scavenging hydroxide and lipid hydroperoxide. Therefore, artifical GPX mimic with high activity may be a promising antioxidant drug, which could prevent or treat the ROS-related diseases.Two artifical GPX mimics, 2-SeCD and 2–TeCD, have been synthesized in our laboratory which exert high activity and good solubility in water. Their GPX actitivties are 7.4 U/μmol and 46.7U/μmol, respectively.In this paper, we focus on the biologic efficacy of these two GPX mimics on the cells level. We investigated the effects of two GPX mimics on the cellular model of Parkinson's disease and atherogenesis in vitro.1. Selenium- containing -cyclodextrin with glutathione peroxidase activity inhibits dopamine-induced apoptosis in rat pheochromocytoma PC12 cellsParkinson's disease (PD) is one of the most common neurological disorders in the elderly individuals, characterized by selective loss of dopaminergic neurons in substantia nigra. Although the cause of PD is still unclear, oxidative stress has been implicated as a pathogenetic factor in the apoptotic cell death of dopaminergic neuron. Accumulating evidence indicates that dopamine (DA), a physiological neurotransmitter, is one of the major sources of reactive oxygen species (ROS). Both auto-oxidation of dopamine and its enzymatic catabolism by monoamine oxidase generate reactive oxygen species (ROS) and is able to induce neuronal cell apoptosis by generation of ROS. Glutathione peroxidase (GPX) is an important antioxidant enzyme in organism and it can quench ROS. 2-selenium-bridged ?-cyclodextrin (2-SeCD) is a GPX mimic generated in our laboratory. Its GPX activity is 7.4 U/μmol.The rat pheochromocytoma cells (PC12) is widely used as a model system for studies of neurodegeneration and Parkinson's disease in vitro. In the present study, we examined whether DA-induced cell death in neuronal PC12 cells could be inhibited by 2-SeCD. Apoptotic cells was detected by annexin V-FITC/PI staining assay and DNA fragmentation was assayed. The content of intracellular reactive oxygen species (ROS) was measured by DCF fluorescence intensity, which was detected by flow cytometry. MDA, a lipid peroxidation product, was also detected. Finally, we examined the levels of Bcl-2 and Bax protein by flow cytometry.Experimental results showed that pretreatment of PC12 with 2-SeCD could attenuate DA-induced cell apoptosis. Moreover, the pretreatment reduced the contents of malondialdehyde (MDA) and ROS in PC12 cells. The pretreatment also increased intracellular GSH content, inhibited the increase of Bax and the decrease of Bcl-2. These results suggested that 2-SeCD was able to attenuate dopamine-induced neural cell apoptosis through suppression of oxidative stress, and this effect were associated with increase of intracellular GSH and modulation of the Bcl-2 family proteins by 2-SeCD.2. Effect of 2–TeCD on the expression of adhesion molecules in human umbilical vein endothelial cells under the stimulation of tumor necrosis factor-a Atherosclerosis is a multifactorial disease characterized by hardening and thickening of the arterial wall. The vascular areas affected by this disease contain mononuclearcells, proliferating smooth muscle cells, and extracellular matrix components. Atherosclerosis is commonly viewed as a chronic inflammatory disease and is associated with certain risk factors such as hyperlipidemia, diabetes, and hypertension. Excessive ROS production has been implicated in the pathogenesis of atherosclerosis. These ROS and/or their modified target biomolecules (ie, oxidized LDL) then serve as true second-messenger coupling molecules to transmit these extracellular signals to elevated expression of atherogenic gene products such as adhesion molecules and other vascular inflammatory gene products. Therefore, this disease may be viewed as an inflammatory disease linked to an abnormality in oxidation-mediated signals in the vasculature.Inflammation plays a major role in the development of atherosclerosis. The recruitment of inflammatory cells from the circulation and their transendothelial migration are critical early event in the development of atherosclerosis. This process is mainly mediated by cell adhesion molecules, such as vascular cell adhesion molecules (VCAM-1) and intercellular adhesion molecules (ICAM-1). Adhesion molecules are produced on the endothelial surface in response to various inflammatory stimuli, like tumor necrosis factor-a (TNF-a), which is commonly found in atherosclerotic lesions. Expression of these adhesion molecules has been shown through a redox-sensitive mechanism involving the redox-regulated transcription factor nuclear factor-kB (NF-κB).The role of cell adhesion molecules in atherosclerosis has been recognized. Therapeutic agents that down-regulate the generation of adhesion molecules will be benefit to atherosclerosis. Since oxidative stress up-regulates the expression of cell adhesion molecules via redox-sensitive transcriptional activation, antioxidants may attenuate expression of adhesion molecule and play an important role in the prevention of atherogenesis. In this paper, we examined the effect of 2-tellurium-bridged ?-cyclodextrin (2–TeCD), a GPx mimic, on the expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs) under tumor necrosis factor-a (TNF-a) stimulation.The expression of ICAM-1 and VCAM-1 on HUVEC surface was detected by indirect immunofluorescence with flow cytometry. Adherence of monocytic cells to endothelial cells was assayed using the Rose Bengal method. The transcription level of ICAM-1 and VCAM-1 mRNAs were detected by RT-PCR Analysis. To determine whether NF-?B was involved in the effect of 2-TeCD on TNF-a-stimulated VCAM-1 and ICAM-1 expression, we detected nuclear p65 to assess NF-?B nuclear translocation by Western blot.Our results indicate that 2–TeCD suppressed the TNF-a-induced the expression of vascular adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) on HUVECs surface in a dose-dependent manner. 2–TeCD also reduced the level of mRNA expression of VCAM-1 and ICAM-1. Furthermore, 2–TeCD inhibited THP-1 monocyte adhesion to HUVECs stimulated by TNF-a. Nuclear factor-?B (NF-?B) could regulate transcription of VCAM-1 and ICAM-1 genes. Western blot analysis showed that 2–TeCD inhibited the translocation of the p65 subunit of NF-?B into the nucleus. Since monocyte adhesion to endothelial cells and subsequent recruitment into the vascular wall is a crucial step in the pathogenesis of atherosclerosis, This property of 2-TeCD may be protective in atherosclerosis.