The Damage Of Human Erythrocyte Membrane Induced By Cigarette Smoke Extract And Inhibited With Tea Catechin

It has been well identified that cigarette smoke contains a varety of toxic chemicals which cause the formation of reactive oxygen species and are associated with increases in the incidence of cardiovascular diseases as well as other human diseases. Aqueous cigarette smoke extract (CSE) is believed to include semiquinone radical and superoxide anion resulting in the production of hydrogen peroxide and hydroxyl radicals. On the other hand, human erythrocyte has been extensively investigated to sustain peroxidative membrane damages in the attack of reactive oxygen species. The erythrocytes under CSE effect would be more prone to alteration of their cellular structure and thus the physiological functions are impaired.In the present study, human erythrocyte was used as an in vitro model to elucidate the CSE damage of erythrocyte at molecular level and to explore the inhibitory effect of epigallocatechin gallate (EGCG), one of the most abundant tea catechins, on the oxidative impairment induced by CSE.CSE was prepared by collecting the gas phase of cigarette smoke and dissolving into phosphate buffer. Erythrocytes separated from whole human blood were incubated with various concentrations of CSE in the absence or presence of EGCG, followed with biochemical assays of melondialdehyde (MDA), methemoglobin (MetHb) and glutathione (GSH) levels, hemolytic rate determination, SDS-PAGE of membrane proteins and chromatographic analysis of membrane phospholipids and fatty acids. The results showed the treatment of erythrocyte with CSE induced hemolysis, a decreased osmotic fragility, depletion of GSH and formation of MDA and MetHb in a dose-dependent manner. Addition of EGCG into the mixtures lowered the changes of these parameters.The SDS-PAGE of ghost membrane showed CSE induced aggregation of membrane proteins, particularly the cytoskeletal proteins. No aggregate was visualized on gel of ghost membrane treated with dithiothreitol (DTT) prior to electrophoresis, indicating the aggregate formation involved in cross-linking of membrane proteins via disulfide bonds. EGCG protected a few membrane proteins from oxidative damage, although had no effect on the formation of high molecular aggregates of proteins, As a mixture of oxidants and other substances triggering biological oxidative stress, CSE attacks the lipid bilayers of cellular membrane and induces alternations of membrane properties including membrane fluidity, enzymatic and trsport activities, and membrane intactness and morphology. High performance liquid chromatographic (HPLC) analyses showed decreases of two major membrane phospholipids phosphatidylcholine and phosphatidylethanolamine of erythrocyte under treatment of CSE. The lowered chromatographic signal at 206 nm could be a consequence of peroxidation and hydrolysis of unsaturated fatty acids of membrane phospholipids. Moreover, CSE had significant influence on fatty acid composition of erythrocyte membrane. Treatment of ghost membrane with CSE caused obvious decreased contents of unsaturated fatty acids such as oleic acid (C18:1), linoleic acid (C18:2), arachidonic acid (C20:4) and tetracosenoic acid (C24:1). In contrast, the relative contents of saturated fatty acids were increased. Pretreating erythrocyte with EGCG protected a few unsaturated fatty acids, for example arachidonic acid from oxidation or peroxidation triggered by CSE, similar to the earlier reports that natural polyphenols played a protective role on membrane lipid bilayers from oxidative stress.In conclusion, CSE induces the oxidative damages of human erythrocytes, which resulted in hemolysis, a decreased osmotic fragility, depletion of GSH, formation of MDA and MetHb, membrane protein aggregation and decomposition of membrane phospholipids and fatty acids. These results confirm the ingredients originated from smoking are able to stress highly biological system with oxidation and peroxidation thus result in the loss of physiological functions of human erythrocytes and other tissues. Tea cetechin EGCG is a natural polyphenol occurred extensively in various teas and other related products. Its antioxidant nature can extert a protective role on the CSE-induced impairment of membrane structure and antioxidant system of erythrocyte. This fact supports that intake of tea catechin-rich food may be helpful in the prevention of smoking and second-hand smoking-induced impairment of physiological functions of erythrocytes and other cell damages, which seems to be related in the lower incidence of smoking related disorders such as lung and cardiovascular diseases, cancer, loss of fertility and age-related diseases.