Study On The Effect Of EGCG On Erythrocyte Membrane

Tea polyphenols are the main functional components of tea leaves, belonging to flavonoids, including flavanols (also known as catechins), flavonols, phenolic acids, depsides, and other polyphenols. Tea catechins, including epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG), are the main biological active substances of tea. Among them EGCG is the most abundant and the most biologically active compound. Tea catechins have been confirmed to be beneficial to human health owing to their polyphenol structure and antioxidant activity. On another hand, however, increasing evidence has demonstrated that EGCG possesses prooxidant potential in biological systems, including modifying proteins, breaking DNA strands and inducing the generation of reactive oxygen species (ROS). In the present study, the prooxidant effects of EGCG on cellular systems were investigated by using human erythrocyte as an in vitro model. SDS-PAGE and NBT-staining assay were utilized to detect the catechol-protein adducts that generated upon treating the cell membranes with EGCG.Experimentals and resultsPart one. The interactions between EGCG and erythrocyte membranes1 EGCG induced the aggregation of erythrocyte membrane proteinsEGCG induced the aggregation of ghost membrane proteins in a dose-dependent manner. Upon incubation of erythrocyte membrane with EGCG, high molecular weight aggregates were generated; and the content of high molecular weight aggregates were increased with increasing concentration of EGCG. Further study with other three catechins (EC, ECG and EGC) indicated that only EGC had same prooxidant effect as EGCG on the erythrocyte membranes, suggesting that the three hydroxyl groups of B-ring structure of catechol is essential for catechins induced aggregation of erythrocyte membrane.2 The effects of metal chelators on the prooxidant activity of EGCGEGCG is easily oxidized or auto-oxidized to form a quinine moiety at either the B-ring or the D-ring through a pathway involving semiquinone radicals and reactive oxygen species. Metal ions such as Fe2+, Fe3+and Cu2+ can play a catalytic role in the free radical-mediated oxidation of EGCG. In the present study, the EGCG-induced high molecular weight aggregates were partially inhibited by EDTA (1 mM). This fact suggests that metal ions play an important role in the EGCG-induced aggregation of erythrocyte membrane proteins, although what kind of specific metal ions involving in this process is still unclear.3 Protein sulfhydryls were involved in the EGCG-induced aggregation of erythrocyte membrane proteinsTo explore the molecular mechanism of the EGCG-induced formation of high molecular weight aggregates (HMWA), thiol reagents and thiol-blocking reagent were co-incubated with the mixture of EGCG and ghost membranes prior to gel electrophoresis. The results showed that thiol-blocking reagent NEM and thiol reagents DTT, Cys and GSH abolished or attenuated the EGCG-induced HMWAs, implicating that protein sulfhydryls were involved in the intermolecular cross-linking of membrane proteins. To further verify the role of protein sulfhydryls in the EGCG-induced aggregation of erythrocyte membrane proteins, the Ellman's method was used to determine the content of protein sulfhydryls in erythrocyte membrane. The results showed that sulfhydryl content was decreased with increasing concentration of EGCG.4 NBT-staining for detecting quinoproteinsBy SDS-PAGE and NBT staining studies have shown that, upon incubating with ghost membranes, EGCG modified membrane proteins with a quinone structure. Thiol reagents NEM and thiol-blocking reagent DTT could completely inhibit the formation of quinoproteins induced by EGCG. Free radical scavengers ascorbic acid can partially inhibit the formation of quinoproteins. Strong staining occurred in the high-molecular-weight area of a gel lane, suggesting that cytoskeletal proteins such as actin and spectrin were able to combine more quinone moieties than other proteins due to their multiple free sulfhydryls.5 The reversibility of the EGCG-induced formation of HMWAsThere is a wealth of evidence showing that the covalent binding between sulfhydryl group and quinone or quinonoid substance is reversible. To explore the reversibility of the EGCG-induced formation of HMWAs, the effects of NEM and DTT on the pre-formed quinone-protein adducts were determined by SDS-PAGE. The results show that both NEM and DTT could partially inhibit the EGCG-induced HMWAs, suggesting that the covalent binding of EGCG with a membrane protein was partly reversible.Part two. The influence of EGCG on erythrocyte1 The influence of EGCG on the content of methemoglobin in erythrocytesIn this work, the effect of EGCG on the composition of Hb/MetHb was investigated in vitro. The results show that 50μM EGCG increased the concentration of MetHb of the cells. This prooxidant effect reached maximum as the concentration of EGCG was more than 100μM. EGCG and NaNO2 together increased the concentration of MetHb of the erythrocytes as the increasing concentration of EGCG.2 The influence of EGCG on the content of GSH in erythrocytesGSH plays important role in the maintenance of normal structure and function of erythrocytes, GSH levels can indirectly reflect the oxidative stress conditions and the antioxidant capacity in red blood cells. Upon incubating with erythrocytes, EGCG and tert-butyl hydroperoxide (t-BHP) didn't significantly change the content of GSH levels.3 The influence of EGCG on the osmotic fragility of erythrocyte membraneIn this experiment, EGCG- and t-BHP-treated erythrocytes were incubated with different concentrations of NaCl solution and the osmotic fragility curve of erythrocyte membrane was plotted. The results showed that the osmotic fragility of the EGCG-treated erythrocytes was not change significantly related to the control samples; the osmotic fragility of t-BHP-treated erythrocytes was increased; while adding EGCG and t-BHP together, it was reduced compared the sample treated with t-BHP alone. It suggested that EGCG is able to play an antioxidant role under oxidative stress.ConclusionEGCG was able to bind covalently to sulfhydryl group of erythrocyte membrane protein, leading to the formation of protein aggregates with intermolecular cross-linking. We suggested that the catechol-quinone originated from the oxidation of EGCG acted as a cross-linker on which peptide chains were combined through thiol-S-alkylation at C2-and C6-sites of the gallyl ring. EGC also induced the formation of protein aggregates, whereas EC and ECG did not, suggesting that a structure with a gallyl moiety is a prerequisite for a catechin to induce the aggragation of membrane proteins. Metal ion-chelator EDTA and free radical-scavenging ascorbic acid inhibited the EGCG-induced aggregation of membrane proteins by blocking the formation of catechol-quinone. In addition, EGCG is also able to play an antioxidant role under oxidative stress.